Methods and compositions for delivery of medicaments to the lungs

ABSTRACT

The disclosure provides a drug composition formulated for inhalation comprising a conjugate of a surface active agent and a pulmonary active drug. The surface active agent has an affinity for the human alveolar/gas interface and comprises at least a portion of a mammalian lung surfactant of a mimic thereof. The disclosure also provides a method of treating a subject suffering from or at risk of suffering from a lung disease comprising administering to the subject a conjugate comprising a drug for lung treatment and a surface active agent by inhalation in an amount effective to induce a drug effect in the lungs.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the national stage of International (PCT) Patent Application Serial No. PCT/US2009/067499, filed Dec. 10, 2009, which claims the benefit of and priority to U.S. provisional patent application Ser. No. 61/121,405, filed Dec. 10, 2008; the entire contents of each application are hereby incorporated by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Sep. 17, 2014, is named GUA-002_SL.txt and is 98,304 bytes in size.

BACKGROUND OF INVENTION

In pulmonary disorders including chronic obstructive pulmonary disease (COPD), chronic bronchitis, and emphysema, there is a chronic obstruction of air flow in and out of the lungs. The obstruction that manifests in these disorders is often permanent and progresses over time. Exacerbations, which are an acute worsening of respiratory function, result in increased morbidity and mortality.

Over the last few decades, research to treat chronic pulmonary disorders such as COPD has focused on identifying inhibitors of human neutrophil elastase (HNE). HNE is a protease capable of degrading numerous proteins including the structural proteins fibronectin, collagen, and elastin. When aberrantly expressed, HNE is one of the most destructive enzymes in the body. HNE is associated with tissue destruction and inflammation and is implicated in numerous pulmonary diseases including COPD, cystic fibrosis, and acute respiratory distress syndrome (ARDS) as well as other diseases of the body. However, the development of HNE inhibitors has been difficult and despite decades of research only one HNE treatment is currently on the market with approval for use only in Japan.

The development of HNE inhibitors and other drugs designed for lung treatment has focused on systemic treatments. A major obstacle with such an approach, whether the drug is delivered orally, parenterally, or by inhalation, is achieving meaningful residence times in the lungs. Thus, there remains an unmet need for effective lung treatments.

SUMMARY OF THE INVENTION

The disclosure provides methods and compositions for delivering medicaments to the lungs. It is now appreciated that a key problem associated with treating lung diseases is the difficulty in obtaining sufficient residence times of active drug molecules in the lungs. The lungs are very adept at clearing foreign matter, such that active drug molecules may be cleared from the lung before the desired medicinal effect is achieved.

Pulmonary surfactants are secreted by Type II pneumocytes in the lungs to reduce surface tension within the alveoli therefore preventing alveolar collapse during expiration. Pulmonary surfactants, which are a complex of lipids and proteins, spread across the alveolar surface to lower surface tension and are maintained in the lung for extended periods. Therefore, the residence time of active drug molecules in the lung can be increased by covalently linking the active drug molecule to a surfactant lipid or protein. Administering active drug molecules covalently linked to a surfactant lipid or protein provides increased duration of action in the lung resulting in substantially fewer doses and better patient compliance, localization of the active drug molecule to the lung resulting in decreased in systemic toxicity, and significantly higher localized lung concentrations for enhanced efficacy.

In one aspect, the invention provides a drug composition, formulated for inhalation, comprising a surface active agent that has an affinity for the human alveolar/gas interface. The surface active agent comprises at least a portion of a mammalian lung surfactant polypeptide or mimic thereof that is substantially non-immunogenic to humans.

The surface active agent is associated with a pulmonary active drug, and preferably covalently bonded to the drug, which binds to an extracellular or cell-surface target or other target accessible to the pulmonary/gas interface. The extracellular or cell-surface target may be, by way of example, an elastase, a TNF receptor, an EGF receptor, an adrenergic receptor, a P2X or P2Y purinergic receptor, or an endothelin receptor. In certain embodiments, the surface active agent covalently bonded to a pulmonary active drug, which binds to an extracellular or cell surface target is administered to a subject suffering from lung disease, including, but not limited to emphysema, chronic bronchitis and acute exacerbation of chronic bronchitis (AECB), chronic obstructive pulmonary disease (COPD), asthma, respiratory distress disorder (RDS), pneumonia (including ventilator associated pneumonia (VAP)), tuberculosis or other bacterial infection, cystic fibrosis, pulmonary arterial hypertension (PAH), and lung cancer.

For example, the intracellular parasite responsible for tuberculosis (TB) is harbored in macrophages. Macrophages normally have a short life ingesting foreign organisms and undergo apoptosis to remove foreign organisms from the body. The TB microorganism corrupts the intracellular suicide communications leaving macrophages essentially immortal, thus permitting the mycoplasm to persist. A signature of immortalized macrophages harboring tuberculosis is the overexpression of P2X purinergic receptor (e.g., P2X7 purinergic receptor) on the macrophage membrane (Placido et al., Cell Immunol. 244:10-8 (2006)). P2X receptor agonists, such as ATP and ATP analogues, will induce apoptosis in these macrophages and thus kill the parasitic TB (Pfeiffer et al., J. Leukoc. Biol. 75:1173 (2004)). Also see, for example: Mokrousov et al., “Mycobacterium Tuberculosis Coexistence with Humans-making an imprint on the macrophage P2X7 gene?,” J. Medical Microbiology, 2008, 57, 581. Benzoyl derivatives of ATP are potent extracellular agonists of P2X receptors (e.g., P2X7 purinergic receptors) and can be covalently linked to the surface active agent and delivered via inhalation as a long duration TB treatment.

ATP and ATP analogues may also be used to agonize P2Y receptors implicated in cystic fibrosis. Cystic fibrosis is a recessive genetic disorder caused by a mutation in the cystic fibrosis transmembrane regulator (CFTR) gene, which encodes a chloride channel. The defective chloride channel results in aberrant ion transport which leads to a reduced mucosal hydration and mucus clearing and ultimately chronic infection of the respiratory tract. P2Y receptors (e.g., P2Y₂ receptor) are expressed on the luminal surface of epithelial cells lining bronchial and lung surfaces. P2Y agonists stimulate chloride secretion, inhibit sodium absorption and enhance airway clearance (Deterding et al., Am. J. Respiratory & Crit. Care Med., 176:362-369 (2007)). Daily administration of an ATP analogue agonizing P2Y receptors provides positive results, but only for a short period of time as systemic side effects arise after a few months of treatment. Covalent linkage of P2Y receptor agonists, such as ATP, ATP analogues, and denufosol tetrasodium, to a surface active agent will increase the residence time of the P2Y agonist in the lungs and reduce the systemic side effects associated with increased levels of ATP in the bloodstream.

Agonists for the β2-adrenergic receptor are well-established bronchodilators for the treatment of asthma (Anderson, Clin. Rev. Allergy Immunol. 31:119-30 (2006). The β2-adrenergic receptor, however, is ubiquitously expressed and thus repeated dosing will likely have deleterious systemic side effects. Covalently linking a β2-adrenergic receptor agonist, for example, albuterol, to a surface active peptide will essentially isolate the active agent to the lung, thus avoiding or diminishing the potential systemic toxicities.

Steriods are also well-established treatments for asthma. The corticosteroid, fluticasone proprionate, is frequently used to treat asthma, but in cases of severe asthma attacks prednisolone is typically more effective. Prednisolone, however, can only be used for a short period of time (e.g., 3-4 days or 2-3 weeks depending on the severity of the asthma attack) due to serious side effects that preclude its chronic use. Covalently linking a steroid, for example, prednisolone, to a surface active agent will increase the residence time of the steroid in the lung converting its use from an acute treatment to a chronic treatment.

Endothelin receptor antagonism is an important therapeutic strategy in PAH, which is an increase in blood pressure in the pulmonary arteries (Leuchte et al., Am. J. Physiol. Lung Cell Mol. Physiol., 2008, 294, L772-L776). PAH is characterized by vasoconstriction and abnormal proliferation of the pulmonary smooth muscle cells. Endothelin receptors A and B are expressed on pulmonary smooth muscle cells and mediate the mitogenic and vasoconstrictive effects of endothlin. Endothlin receptor antagonists, such as bosentan and sitaxentan, induce vasodilation, block the abnormal proliferation of smooth muscle cells, and have been shown to improve the pulmonary hemodynamics of patients with PAH. However, administration of endothlin receptor antagonists also results in serious side effects such as hepatocellular injury. Covalently linking an endothlin receptor antagonist, for example, bosentan, to a surface active agent will isolate the active agent in the lung thus reducing systemic toxicities. Other drugs used in the treatment of PAH include prostacyclin analogues and phosphodiesterase type 5 (PDE5) inhibitors. In particular, most prostacyclin analogues, for example, epoprostenol, are administered by continuous infusion or by injection, which can be painful and dangerous. Covalently linking a prostacyclin analogue to a surface active agent coupled with inhalation delivery will have the advantage of selective deposition in the lungs with less systemic side effects and potentially better patient compliance.

The epidermal growth factor receptor (EGFR) has been validated as an anticancer target (Carney, Expert Rev. Mol. Diagn. 7:309-19 (2007)) with products such as herceptin on the market to treat breast cancer. Inhibiting the activation of EGFR is a potential treatment for nonsmall cell lung cancer (Y. H. Ling et al., Molecular Pharmacology 72:248-58 (2007)). One hurdle in targeting the EGFR family (which includes HER1, HER2, HER3, and HER4) is that when one receptor is inactivated that the other receptors compensate for the inactivated receptor. In addition, there are eleven known endogenous ligands for the EGFR family. Matrix metalloproteinase (e.g., ADAM10 and ADAM17, also known as sheddases) have been shown to release endogenous ligands of EGFR via a cell surface shedding mechanism (Horiuchi et al., Mol. Biol. Cell 18:176-188 (2007); Gazdar & Minna, Cancer Prey Res, 1(3):156-160 (2008); Zhou et al., Cancer Cell, 10:39-50 (2006)). Non-selective inhibitors of matrix metalloproteinase enzymes have been identified, however, ubiquitous inhibition of this enzyme class will likely have deleterious systemic side effects. Covalently linking a non-selective inhibitor of matrix metalloproteinase (e.g., marimastat) to a surface active agent will isolate the active agent to the lungs and deactivate all four EGFR pathways in the lungs.

These examples including emphysema, tuberculosis, cystic fibrosis, asthma, pulmonary arterial hypertension (PAH), and nonsmall cell lung cancer demonstrate the generality of covalently linking therapeutic molecules to agents that preferentially reside in the lung with inhalation delivery.

In another embodiment, the surface active agent is covalently bonded to a pulmonary active drug and a cell membrane permeable transport molecule that enters the lung cells. Agents that function within the cell include, but are not limited to retinoids, survivin inhibitors, and caspase promoters.

In another embodiment, the surface active agent comprises a human lung surfactant or a non-human mammalian lung surfactant or a fraction thereof. Exemplary non-human mammalian lung surfactants include bovine, porcine, or ovine lung surfactants or a fraction thereof. The agent may comprise or be derived from a mammalian lung surfactant harvested from the lungs of a human or non-human mammal.

In another embodiment, the surface active agent comprises at least a portion of a mammalian lung surfactant polypeptide, an allelic variant thereof, or a synthetic mimic thereof. The agent may comprise a natural surfactant polypeptide, such as SP-A, SP-B, SP-C, SP-D, portions thereof, or mixtures thereof. The agent may comprise a mixture of SP-A, SP-B, SP-C, SP-D or portions thereof. Exemplary peptides include at least about a 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acid fragment of a natural surfactant polypeptide. The surface active agent may comprise at least a portion of SP-B. Exemplary SP-B polypeptides include at least about a 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acid fragment of SP-B. An SP-B peptide may be an amino-terminal peptide or a carboxy-terminal peptide. An exemplary SP-B peptide may be a 25-amino acid amino terminal peptide.

In another embodiment, the surface active agent comprises a synthetically produced peptide. A peptidomimetic may comprise at least one deletion or amino acid substitution mutant of a mammalian lung surfactant polypeptide. A peptidomimetic may comprise at least one deletion or amino acid substitution mutant of a human lung surfactant polypeptide.

In another embodiment, the surface active agent may comprise a surfactant polypeptide that is recombinantly produced. A recombinant mammalian lung surfactant polypeptide, such as SP-A, SP-B, SP-C, SP-D, or a portion thereof may be produced by expressing the DNA coding for SP-A, SP-B, SP-C, SP-D, or a portion thereof in a prokaryotic or eukaryotic expression system. Recombinant surfactant polypeptides may be the same or differ from mammalian lung surfactant polypeptides. A recombinant polypeptide may comprise at least one deletion or amino acid substitution mutant of a mammalian, preferably a human lung surfactant polypeptide.

In another embodiment, the surface active agent comprises both a surfactant polypeptide and a lipid.

The surface active agent preferably is covalently bonded to a pulmonary active drug. The pulmonary active drug may be covalently linked to a surfactant protein or lipid. The pulmonary active drug may be covalently bonded to an amino- or carboxy-terminal amino acid or an internal amino acid of a surfactant polypeptide. In certain embodiments, more than one pulmonary active drug is bound to a surface active agent. In other embodiments, a single pulmonary active drug is bound to a surface active agent and mixed with at least one other pulmonary active drug bound to a surface active agent.

In one embodiment, the a pulmonary active drug molecule is extended with an amino acid or mimetic linker, such as a glycine linker, to create an unnatural amino acid that can be used in automatic peptide synthesis. The extended molecule (i.e., the drug plus the amino acid linker) can then be attached to the surface active agent through an amino- or hydroxyl-group.

The pulmonary active drug binds to an extracellular or cell-surface bound target that is accessible to the pulmonary/gas interface. Generally, the pulmonary active drug portion of the conjugate may be any drug that has utility in the management or treatment of lung disease and, preferably are those characterized by a molecular weight below about 10,000 KD. Such small molecule drugs, as opposed to high molecular weight biologics, are particularly well-suited for delivery by inhalation in accordance with the teachings of this invention. Indeed, many drug moieties having a molecular weight below about 5,000 KD, or about 2000 KD may be used. In certain embodiments, the pulmonary active drug may be an elastase inhibitor, corticosteroid, bronchodilator, antihistamine, antibiotic, P2X agonist, P2Y agonist, chemotherapeutic agent, matrix metalloproteinase inhibitor, ADAM inhibitor, kinase inhibitor, endothelin receptor antagonist, prostacyclin analogue, or phosphodiesterase type 5 inhibitor.

In other embodiments, the pulmonary active drug may be a polypeptide such as an antibody (e.g., an intact antibody, an antigen binding fragment such as a Fab, Fab′, (Fab′)₂, Fv fragment, or a biosynthetic antibody such as a single Fv or SFv molecule) directed against any targets disclosed herein or addressed by the drugs disclosed herein, a fusion protein or a peptidomimetic.

In certain embodiments, more than one pulmonary active drug may be covalently bonded to a surface active agent and administered in combination. When more than one pulmonary active drug is covalently bonded to a surface active agent, the drug may be the same drug, a member of the same drug class, or a member of a different drug class.

The drug composition is delivered to the lungs of a human patient by an inhalation device. Exemplary inhalation devices include fixed dose inhalers, metered dose inhalers, and nebulizers.

In one embodiment, the invention provides a drug composition formulated for inhalation. The drug composition comprises a surface active agent characterized by an affinity for the human alveolar/gas interface, wherein the surface active agent comprises at least a portion of a mammalian lung surfactant polypeptide or a mimic thereof, and covalently bonded to said agent is a pulmonary active drug which binds to an extracellular or cell surface-bound target accessible to the pulmonary/gas interface. The pulmonary active drug may be an antihistamine, a P2Y agonist, a matrix metalloproteinase inhibitor, an ADAM inhibitor, a kinase inhibitor, an endothelin receptor antagonist, a prostacyclin analogue, and a phophodiesterase type 5 inhibitor.

In another aspect, the invention provides a method for treating a subject suffering from or at risk of a lung disease. The method comprises administering a conjugate comprising a pulmonary active drug covalently bonded to a surface active agent characterized by an affinity for the human alveolar/gas interface, wherein the surface active agent comprises at least a portion of a mammalian lung surfactant polypeptide or a mimic thereof that is substantially non-immunogenic to humans. The conjugate is administered to the subject by inhalation in an amount effective to induce a drug effect in the lungs.

The method of administration targets the pulmonary active drug to the lungs of a subject in need thereof. The modified compositions of matter and the methods of administration have the dual advantage that they reduce the systemic bioavailability and distribution of the pulmonary active drug relative to inhalation administration of an unconjugated drug, and this provides an advantage in dealing with off target toxicities. The modified compositions of matter and the methods of administration also increase the residence time of the drug in the lung relative to inhalation administration of an unconjugated drug, and therefore very significantly improve lung bioavailability. The result can be a reformulation or redesign of a drug that has unacceptable toxicity and/or poor efficacy, even though it binds and inhibits the target well to produce a clinically valuable modified drug.

The method of administration targets the pulmonary active drug to the lungs of a subject in need thereof. The method of administration reduces the systemic bioavailability of the drug relative to inhalation administration of an unconjugated drug. The method of administration increases the residence time of the drug in the lung relative to inhalation administration of an unconjugated drug.

In one embodiment, the administration of a pulmonary active drug-surface active agent conjugate reduces the dosing frequency relative to administration of an unconjugated drug. The administration step may be repeated once daily, every other day, every three days, every four days, every five days, biweekly, or weekly. The administration step may be conducted using an inhaler, an aerosol, particulates with or without propellants, metered dosages, or a nebulizer.

In certain embodiments, the subject in need of treatment is suffering from lung inflammation or disease or is at risk of suffering from a lung disease. The subject in need of treatment may be suffering from emphysema, chronic bronchitis and acute exacerbation of chronic bronchitis (AECB), chronic obstructive pulmonary disease (COPD), asthma, respiratory distress disorder (RDS), pneumonia (including ventilator associated pneumonia (VAP)), tuberculosis or other bacterial infection, cystic fibrosis, pulmonary arterial hypertension (PAH), and/or lung cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the nucleic acid sequence that encodes human surfactant protein A (SEQ ID NO: 1). FIG. 1B shows the amino acid sequence for human surfactant protein A (SEQ ID NO: 2).

FIG. 2A shows the nucleic acid sequence that encodes human surfactant protein B (SEQ ID NO: 3). FIG. 2B shows the amino acid sequence for human surfactant protein B (SEQ ID NO: 4). FIG. 2C shows the amino acid sequence for mature human surfactant protein B (SEQ ID NO: 5).

FIG. 3A shows the nucleic acid sequence that encodes human surfactant protein C (SEQ ID NO: 6). FIG. 3B shows the amino acid sequence for human surfactant protein C (SEQ ID NO: 7). FIG. 3C shows the amino acid sequence for mature human surfactant protein C (SEQ ID NO: 8).

FIG. 4A shows the nucleic acid sequence that encodes human surfactant protein D (SEQ ID NO: 9). FIG. 4B shows the amino acid sequence for human surfactant protein D (SEQ ID NO: 10). FIG. 4C shows the amino acid sequence for mature human surfactant protein D (SEQ ID NO: 11).

FIG. 5A shows the nucleic acid sequence that encodes bovine surfactant protein A (SEQ ID NO: 12). FIG. 5B shows the amino acid sequence for bovine surfactant protein A (SEQ ID NO: 13).

FIG. 6A shows the nucleic acid sequence that encodes bovine surfactant protein B (SEQ ID NO: 14). FIG. 6B shows the amino acid sequence for bovine surfactant protein B (SEQ ID NO: 15).

FIG. 7A shows the nucleic acid sequence that encodes bovine surfactant protein C (SEQ ID NO: 16). FIG. 7B shows the amino acid sequence for bovine surfactant protein C (SEQ ID NO: 17).

FIG. 8A shows the nucleic acid sequence that encodes bovine surfactant protein D (SEQ ID NO: 18). FIG. 8B shows the amino acid sequence for bovine surfactant protein D (SEQ ID NO: 19).

FIG. 9A shows the nucleic acid sequence that encodes porcine surfactant protein A (SEQ ID NO: 20). FIG. 9B shows the amino acid sequence for porcine surfactant protein A (SEQ ID NO: 21).

FIG. 10A shows the nucleic acid sequence that encodes a partial porcine surfactant protein B (SEQ ID NO: 22). FIG. 10B shows a partial amino acid sequence for porcine surfactant protein B (SEQ ID NO: 23).

FIG. 11A shows the nucleic acid sequence that encodes porcine surfactant protein C (SEQ ID NO: 24). FIG. 11B shows the amino acid sequence for porcine surfactant protein C (SEQ ID NO: 25).

FIG. 12A shows the nucleic acid sequence that encodes porcine surfactant protein D (SEQ ID NO: 26). FIG. 12B shows the amino acid sequence for porcine surfactant protein D (SEQ ID NO: 27).

FIG. 13A shows the nucleic acid sequence that encodes ovine surfactant protein A (SEQ ID NO: 28). FIG. 13B shows the amino acid sequence for ovine surfactant protein A (SEQ ID NO: 29).

FIG. 14A shows the nucleic acid sequence that encodes ovine surfactant protein B (SEQ ID NO: 30). FIG. 14B shows the amino acid sequence for ovine surfactant protein B (SEQ ID NO: 31).

FIG. 15A shows the nucleic acid sequence that encodes ovine surfactant protein C (SEQ ID NO: 32). FIG. 15B shows the amino acid sequence for ovine surfactant protein C (SEQ ID NO: 33).

FIG. 16A shows the nucleic acid sequence that encodes a partial ovine surfactant protein D (SEQ ID NO: 34). FIG. 16B shows a partial amino acid sequence for ovine surfactant protein D (SEQ ID NO: 35).

FIG. 17 is a table depicting exemplary human neutrophil elastase (HNE) inhibitors. The reference numbers listed in the table correspond to the compound identifiers referred to in Philip D. Edwards and Peter R. Bernstein in “Synthetic Inhibitors of Elastase,” Medicinal Research Reviews, Vol. 14, No. 2, 127-194 (1994).

FIG. 18 is a schematic diagram depicting the chemical synthesis of a representative emphysema inhibitor. The emphysema inhibitor as shown contains a glycine linker. The glycine linker (circled) converts the compound to an unnatural amino acid that may be used in a standard peptide synthesis reaction for covalent coupling to the N-terminal 1-25 amino acids of SP-B.

FIG. 19 is a table depicting exemplary targets for use as HNE inhibitors. Target 2 attached to the N-terminus of the first 25 residues of the human surfactant B peptide forms target C. Similarly, target 3 attached to the N-terminus of the first 25 residues of the human surfactant B peptide forms target B.

FIG. 20 is a schematic diagram depicting the chemical structures of antihistamine drugs. Circled side chains depict exemplary positions where a lung surfactant moiety may be linked.

FIG. 21 is a schematic diagram depicting the chemical structure of marimastat, a nonselective matrix metalloproteinase inhibitor, derivatized with glycine linker.

FIG. 22 provides photographs (A-D) of lung histology sections from four mice with HNE induced emphysema.

FIG. 23 shows photographs (A-D) of lung histology sections from four mice with HNE induced emphysema treated with a Zeneca inhibitor (target 2).

FIG. 24 shows photographs (A-D) of lung histology sections from four mice from four mice instilled with saline.

FIG. 25 shows photographs (A-D) of lung histology sections from four mice with HNE induced emphysema treated with a Zeneca HNE inhibitor linked to a lung surfactant B peptide (target C).

DETAILED DESCRIPTION OF THE INVENTION

Surfactant Proteins

The surface active agent comprises at least a portion of a mammalian lung surfactant polypeptide that is substantially non-immunogenic to humans. The polypeptide or portion thereof may be a mammalian lung surfactant moiety or a synthetic mimic thereof. Exemplary surfactant polypeptides may be animal-derived, recombinant, synthetic, analogs, or peptide mimetics.

Natural lung surfactant proteins include SP-A, SP-B, SP-C, SP-D, or portions thereof, alone or in combination with lipids (U.S. Pat. No. 5,302,581). In some embodiments, the surface active agent comprises the full length surfactant polypeptide. In other embodiments, the surface active agent comprises a portion of a surfactant polypeptide. For example, human SP-B is a 79 amino acid residue polypeptide, however, the N-terminal 25 amino acid residues of SP-B possess therapeutic effects comparable to the whole peptide (Kurutz and Lee, Biochem., 41, 9627-36 (2002)). Exemplary peptides of natural lung surfactant proteins may be at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length. Exemplary peptides of human SP-B are shown in Table 1.

In one embodiment, the surface active agent comprises human lung surfactant obtained by lung lavage of human cadavers at autopsy or by lung lavage of consenting adults.

In certain embodiments, the surface active agent comprises a non-human mammalian lung surfactant or a fraction thereof. Exemplary non-human surfactants include bovine, porcine or ovine lung surfactants or a fraction thereof. The non-human surfactant may be harvested from the lungs of a non-human mammal using techniques that are well known in the art. For example, porcine surfactant may be obtained from newborn and/or adult pigs harvesting the bronchoalveolar lavage (BAL) of the lungs with saline as described in Bernhard et al., Am. J. Respir. Cell Mol. Biol. 17:41-50 (1997), which is incorporated herein by reference. Harvested BAL fluid is centrifuged to remove cells and then the cell-free BAL fluid is further centrifuged to generate a raw surfactant pellet. Ovine surfactant may be obtained from whole lung lavages of adult sheep as described by Brackenbury et al., Am. J. Respir Cir. Care Med. 163:1135-1142 (2001), which is incorporated herein by reference. The harvested alveolar lavage is centrifuged to remove cellular debris, followed by further centrifugation to obtain a pellet corresponding to a surfactant aggregate pellet. Bovine surfactant may also be obtained from the lung lavages of adult cows as described by Panda et al. (J Colloid Interface Sci., 311:551-5 (2007)), which is incorporated herein by reference. Alveofact®, a natural bovine surfactant extract containing phospholipids, neutral lipids, SP-B and SP-C polypeptides may also be used.

Proteins and polypeptides derived from or having characteristics similar to those human lung surfactant may also be used. For example, SP-B may be isolated from bovine surfactant using differential organic extraction, column chromatography, and/or preparative SDS-PAGE as described by Beers et al., Am. J. Physiol Lung Cell Mol. Physiol. 262:L773-L778 (1992), which is incorporated herein by reference.

The mammalian lung surfactant polypeptides or portion thereof can also be recombinantly produced. Recombinant SP-A, SP-B, SP-C, SP-D, or a portion thereof is obtainable by expression of a DNA sequence coding for SP-A, SP-B, SP-C, SP-D, or a portion thereof in a suitable prokaryotic or eukaryotic expression system using various known techniques. Recombinant vectors, which are readily adapted to include a isolated nucleic acid encoding a surfactant polypeptide or a portion thereof, host cells containing the recombinant vectors, and methods of making such vectors and host cells as well as using them for the production of the encoded polypeptides by recombinant techniques are well-known. The nucleic acids encoding a surfactant polypeptide or a portion thereof may be provided in an expression vector comprising a nucleotide sequence encoding a surfactant polypeptide that is operably linked to at least one regulatory sequence. It should be understood that the design of the expression vector may depend on such factors as the choice of the host cell to be transformed and/or the type of protein desired to be expressed. The vector copy number, the ability to control that copy number, and the expression of any other protein encoded by the vector, such as antibiotic markers, should be considered. The subject nucleic acids may be used to cause expression and over-expression of a kinase or phosphatase polypeptide in cells propagated in culture, e.g., to produce proteins or polypeptides, including fusion proteins or polypeptides.

Host cells may be transfected with a recombinant gene in order to express a surfactant polypeptide or portion thereof. The host cell may be any prokaryotic or eukaryotic cell. For example, a polypeptide may be expressed in bacterial cells, such as E. coli, insect cells (baculovirus), yeast, or mammalian cells. In those instances when the host cell is human, it may or may not be in a live subject. Other suitable host cells are known to those skilled in the art. Additionally, the host cell may be supplemented with tRNA molecules not typically found in the host so as to optimize expression of the polypeptide. Other methods suitable for maximizing expression of the polypeptide will be known to those in the art.

Methods of producing polypeptides are well-known in the art. For example, a host cell transfected with an expression vector encoding a surfactant polypeptide or portion thereof may be cultured under appropriate conditions to allow expression of the polypeptide to occur. The polypeptide may be secreted and isolated from a mixture of cells and medium containing the polypeptide. Alternatively, the polypeptide may be retained cytoplasmically. Cells are then harvested, lysed, and the protein is isolated from the cell lysates.

A cell culture includes host cells, media, and other by-products. Suitable media for cell culture are well known in the art. The polypeptide may be isolated from cell culture medium, host cells, or both using techniques known in the art for purifying proteins, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, gel filtration chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, lectin chromatography, ultrafiltration, electrophoresis, immunoaffinity purification with antibodies specific for particular epitopes of a polypeptide of the invention, and high performance liquid chromatography (“HPLC”) is employed for purification. Thus, a nucleotide sequence encoding all or a selected portion of a surfactant polypeptide may be used to produce a recombinant form of the protein via microbial or eukaryotic cellular processes. Ligating the sequence into a polynucleotide construct, such as an expression vector, and transforming or transfecting into hosts, either eukaryotic (yeast, avian, insect or mammalian) or prokaryotic (bacterial cells), are standard procedures. Similar procedures, or modifications thereof, may be employed to prepare recombinant polypeptides of the invention by microbial means or tissue-culture technology.

Expression vehicles for production of a recombinant protein include plasmids and other vectors. For instance, suitable vectors for the expression of a polypeptide of the invention include plasmids of the types: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmids for expression in prokaryotic cells, such as E. coli.

In certain embodiments, mammalian expression vectors contain both prokaryotic sequences to facilitate the propagation of the vector in bacteria, and one or more eukaryotic transcription units that are expressed in eukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples of mammalian expression vectors suitable for transfection of eukaryotic cells. Some of these vectors are modified with sequences from bacterial plasmids, such as pBR322, to facilitate replication and drug resistance selection in both prokaryotic and eukaryotic cells. Alternatively, derivatives of viruses such as the bovine papilloma virus (BPV-I), or Epstein-Barr virus (pHEBo, pREP-derived and p205) can be used for transient expression of proteins in eukaryotic cells. The various methods employed in the preparation of the plasmids and transformation of host organisms are well known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells, as well as general recombinant procedures, see Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press, 1989) Chapters 16 and 17. In some instances, it may be desirable to express the recombinant protein by the use of a baculovirus expression system. Examples of such baculovirus expression systems include pVL-derived vectors (such as pVL1392, pVL1393 and pVL941), pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derived vectors (such as the [beta]-gal containing pBlueBac III).

In another embodiment, protein production may be achieved using in vitro translation systems. In vitro translation systems are, generally, a translation system which is a cell-free extract containing at least the minimum elements necessary for translation of an RNA molecule into a protein. An in vitro translation system typically comprises at least ribosomes, tRNAs, initiator methionyl-tRNAMet, proteins or complexes involved in translation, e.g., eIF2, eIF3, the cap-binding (CB) complex, comprising the cap-binding protein (CBP) and eukaryotic initiation factor 4F (eIF4F). A variety of in vitro translation systems are well known in the art and include commercially available kits. Examples of in vitro translation systems include eukaryotic lysates, such as rabbit reticulocyte lysates, rabbit oocyte lysates, human cell lysates, insect cell lysates and wheat germ extracts. Lysates are commercially available from manufacturers such as Promega Corp., Madison, Wis.; Stratagene, La Jolla, Calif.; Amersham, Arlington Heights, IU.; and GIBCO/BRL, Grand Island, N.Y. In vitro translation systems typically comprise macromolecules, such as enzymes, translation, initiation and elongation factors, chemical reagents, and ribosomes. In addition, an in vitro transcription system may be used. Such systems typically comprise at least an RNA polymerase holoenzyme, ribonucleotides and any necessary transcription initiation, elongation and termination factors. In vitro transcription and translation may be coupled in a one-pot reaction to produce proteins from one or more isolated DNAs. When expression of a carboxy terminal fragment of a polypeptide is desired, i.e., a truncation mutant, it may be necessary to add a start codon (ATG) to the oligonucleotide fragment containing the desired sequence to be expressed. It is well known in the art that a methionine at the N-terminal position may be enzymatically cleaved by the use of the enzyme methionine aminopeptidase (MAP). MAP has been cloned from E. coli (Ben-Bassat et al., (1987) J Bacteriol. 169:751-757) and Salmonella typhimurium and its in vitro activity has been demonstrated on recombinant proteins (Miller et al., (1987) PNAS USA 54:2718-1722). Therefore, removal of an N-terminal methionine, if desired, may be achieved either in vivo by expressing such recombinant polypeptides in a host which produces MAP (e.g., E. coli or CM89 or S. cerevisiae), or in vitro by use of purified MAP (e.g., procedure of Miller et al).

Polypeptides of the invention may also be subject to various changes, such as insertions, deletions, and substitutions, either conservative or non-conservative, where such changes provide for certain advantages in their use. Conservative substitutions are those in which one amino acid residue is replaced by another, biologically similar residue. Examples of conservative substitutions include the substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another such as between arginine and lysine, between glutamic and aspartic acids or between glutamine and asparagine and the like. The term “conservative substitution” also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid provided that such a polypeptide also displays the requisite binding activity.

Polypeptides of the invention may also be truncated relative to the full-length mature polypeptide. Polypeptides may be truncated at either the amino-terminus, carboxy-terminus, or both termini. Polypeptides may be truncated by at least one amino acid, or at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or 70 amino acids.

A mammalian lung surfactant polypeptide or a portion thereof can be synthesized from amino acids by techniques that are known to those skilled in the polypeptide art. A summary of the many techniques available may be found in J. M. Steward and J. D. Young, “Solid Phase Peptide Synthesis”, W.H. Freeman Co., San Francisco, 1969, and J. Meienhofer, “Hormonal Proteins and Peptides”, Vol. 2, p. 46, Academic Press (New York), 1983 for solid phase peptide synthesis, and E. Schroder and K. Kubke, “The Peptides”, Vol. 1, Academic Press (New York), 1965 for classical solution synthesis.

In general, these methods comprise the sequential addition of one or more amino acid residues or suitably protected amino acid residues to a growing peptide chain. Normally, either the amino or carboxyl group of the first amino acid residue is protected by a suitable, selectively removable protecting group. A different, selectively removable protecting group is utilized for amino acids containing a reactive side group (e.g., lysine).

Using a solid phase synthesis as an example, the protected or derivatized amino acid is attached to an inert solid support through its unprotected carboxyl or amino group. The protecting group of the amino or carboxyl group is then selectively removed and the next amino acid in the sequence having the complementary (amino or carboxyl) group suitably protected is admixed and reacted under conditions suitable for forming the amide linkage with the residue already attached to the solid support. The protecting group of the amino or carboxyl group is then removed from this newly added amino acid residue, and the next amino acid (suitably protected) is then added, and so forth. After all the desired amino acids have been linked in the proper sequence, any remaining terminal and side group protecting groups (and any solid support) are removed sequentially or concurrently, to afford the final polypeptide. That polypeptide is then washed by dissolving in a lower aliphatic alcohol, and dried. The dried surfactant polypeptide can be further purified by known techniques, if desired.

In certain embodiments, commonly used methods such as t-BOC or f-MOC protection of alpha-amino groups can be used. Both methods involve stepwise syntheses whereby a single amino acid is added at each step starting from the C-terminus of the peptide (See, Coligan et al., Current Protocols in Immunology, Wiley Interscience, 1991, Unit 9). Peptides of the invention can be synthesized, for example, by the well known solid phase peptide synthesis methods described in Merrifield, J. Am. Chem. Soc. 85: 2149, 1962, and Stewart & Young, 1969, Solid Phase Peptides Synthesis, pp. 27-62, using a copoly(styrene-divinylbenzene) containing 0.1-1.0 mMol amines/g polymer. On completion of chemical synthesis, the peptides can be deprotected and cleaved from the polymer by treatment with liquid HF-10% anisole for about ¼-1 hours at 0° C. After evaporation of the reagents, the peptides are extracted from the polymer with 1% acetic acid solution which is then lyophilized to yield the crude material. This can normally be purified by such techniques as gel filtration on Sephadex G-15 using 5% acetic acid as a solvent. Lyophilization of appropriate fractions of the column will yield the homogeneous peptide or peptide derivatives, which can then be characterized by such standard techniques as amino acid analysis, thin layer chromatography, high performance liquid chromatography, ultraviolet absorption spectroscopy, molar rotation, solubility, and quantitated by the solid phase Edman degradation.

In one embodiment, recombinant and/or synthetic SP-B peptides contain amino acids 2, 4, 6, and 9 of SEQ ID NO:5. Prolines 2, 4, and 6 and tryptophan 9 of SEQ ID NO:5 may constitute essential structural motifs for protein function. In some embodiments, SP-B peptides may be substituted at any amino acid residue other than tryptophan 9 amino acid (relative to SEQ ID NO:5).

A lung surfactant polypeptide mimic is generally a polypeptide that is engineered to mimic the essential attributes of human surfactant protein. An exemplary mimetic peptide mimics SP-B. One example of a SP-B mimic is KL4, a 21 amino acid residue peptide comprising the sequence KLLLLKLLLLKLLLLKLLLLK (SEQ ID NO: 94). This SP-B mimetic protein is also known as Lucinactant (Surfaxin®, Discovery Laboratories).

Surfactant Lipids

In certain embodiments, a surface active agent for use in the invention comprises a surfactant protein, a portion thereof, or a mixture thereof, which associates with natural surfactant lipids in vivo. In other embodiments, a surface active agent for use in the invention comprises a lipid or a lipid-protein complex.

Natural mammalian lung surfactant is a complex of phospholipids, neutral phospholipids, and proteins. Surface active agent for use in the invention disclosed herein may comprise one or more lipids. In some embodiments, the surface active agent can comprise, for example, from as little as about 0.05 to 100% weight percent lipid, so long as the resulting composition has surfactant activity. By weight percent is meant the percentage of a compound by weight in a composition by weight. Thus, a composition having 50 weight percent lipid contains, for example, 50 grams lipids per 100 grams total composition. A surface active agent may contain 0.1 to 50 weight percent lipid, although higher concentrations of lipid can be used. Surface active agents containing both phospholipid and a surfactant polypeptide or portion thereof can contain, therefore, 0.1, 1, 10, 50, 80, to almost 100 weight percent lipid and about 50, 20, 10, to less than 1 weight percent surfactant polypeptide. Alternatively, surface active agents may contain the reverse ratios of lipid to surfactant polypeptide.

The term “lipid” as used herein refers to a naturally occurring, synthetic or semi-synthetic (i.e., modified natural) compound which is generally amphipathic. The lipids typically comprise a hydrophilic component and a hydrophobic component. Exemplary lipids include, but are not limited, phospholipids, fatty acids, fatty alcohols, neutral fats, phosphatides, oils, glycolipids, aliphatic alcohols, waxes, terpenes and steroids. The phrase semi-synthetic (or modified natural) denotes a natural compound that has been chemically modified in some fashion.

Examples of phospholipids include native and/or synthetic phospholipids. Phospholipids that can be used include, but are not limited to, phosphatidylcholines (saturated and unsaturated), phospatidylglycerols, phosphatidylethanolamines, phosphatidylserines, phosphatidic acids, phosphatidylinositols, sphingolipids, diacylglycerides, cardiolipin, ceramides, cerebrosides and the like. Exemplary phospholipids include, but are not limited to, dipalmitoyl phosphatidylcholine (DPPC), dilauryl phosphatidylcholine (DLPC) (C12:0), dimyristoyl phosphatidylcholine (DMPC) (C14:0), distearoyl phosphatidylcholine (DSPC), diphytanoyl phosphatidylcholine, nonadecanoyl phosphatidylcholine, arachidoyl phosphatidylcholine, dioleoyl phosphatidylcholine (DOPC) (C18:1), dipalmitoleoyl phosphatidylcholine (C16:1), linoleoyl phosphatidylcholine (C18:2), myristoyl palmitoyl phosphatidylcholine (MPPC), steroyl myristoyl phosphatidylcholine (SMPC), steroyl palmitoyl phosphatidylcholine (SPPC), palmitoyloleoyl phosphatidylcholine (POPC), palmitoyl palmitooleoyl phosphatidylcholine (PPoPC), dipalmitoyl phosphatidylethanolamine (DPPE), palmitoyloleoyl phosphatidylethanolamine (POPE), dioleoylphosphatidylethanolamine (DOPE), dimyristoyl phosphatidylethanolamine (DMPE), distearoyl phosphatidylethanolamine (DSPE), dioleoyl phosphatidylglycerol (DOPG), palmitoyloleoyl phosphatidylglycerol (POPG), dipalmitoyl phosphatidylglycerol (DPPG), dimyristoyl phosphatidylglycerol (DMPG), distearoyl phosphatidylglycerol (DSPG), dimyristoylphosphatidylserine (DMPS), distearoylphosphatidylserine (DSPS), palmitoyloleoyl phosphatidylserine (POPS), soybean lecithin, egg yolk lecithin, sphingomyelin, phosphatidylinositols, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidic acids, and egg phosphatidylcholine (EPC).

Examples of fatty acids and fatty alcohols include, but are not limited to, sterols, palmitic acid, cetyl alcohol, lauric acid, myristic acid, stearic acid, phytanic acid, dipamlitic acid, and the like. Exemplary fatty acids include palmitic acid.

Examples of fatty acid esters include, but are not limited to, methyl palmitate, ethyl palmitate, isopropyl palmitate, cholesteryl palmitate, palmityl palmitate sodium palmitate, potassium palmitate, tripalmitin, and the like.

Surfactant polypeptide and surfactant lipids interact by hydrostatic interactions. Charged amino acids interact with the lipid polar head groups and hydrophobic amino acids interact with phospholipid acyl side chains. For example, SP-B and SP-C are hydrophobic proteins. Both SP-B and SP-C preferentially bind anionic lipids, such as phosphatidylglycerol (PG), and not DPPC. SP-A and SP-D are hydrophilic proteins and interact with a broad range of amphipathic lipids, including glycerophospholipids, sphingophospholipids, glycosphingolipids, lipid A, and lipoglycans. SP-A binds DPPC. By way of example, hydrostatic interactions are observed with the SP-B mimetic, KL4, and lipids in natural surfactant or lipids comprised in the surface active agent. For example, the lysine residues in the KL4 peptide interact with the charge head groups of DPPC and the hydrophobic leucine resides interact with the phospholipid acyl side chains of phosphatidylglycerol.

In certain embodiments, a drug composition as disclosed herein comprises a surface active agent comprising a portion of a mammalian lung surfactant polypeptide or mimic thereof and does not additionally comprise a lipid or a mixture of lipids. Drug compositions administered by inhalation comprising surface active agents comprising only a portion of a mammalian lung surfactant polypeptide or mimic thereof can interact with natural surfactant in the lungs through hydrostatic interactions. For example, recombinant SP-B can interact with natural surfactant in the lungs by binding anionic phospholipids, such as phosphatidylglycerol.

In other embodiments, a drug composition as disclosed herein comprises a surface active agent comprising both a portion of a mammalian lung surfactant polypeptide or a mimic thereof and at least one lipid. To facilitate absorption of drug compositions comprising both a polypeptide or mimic thereof and at least one lipid into natural surfactant in the lungs, phopholipid monolayers mimicking those found in natural surfactant can be used. Exemplary lipid mixtures include dipalmitoylphosphatidylcholine/palmitoyloleoylphosphatidylglycerol, for example at a 7:3 w/w ratio. The mammalian lung surfactant polypeptide can be inserted into the phosphoplipid monolayer and the protein/lipid mix can be absorbed into the natural surfactant at the alveolar/gas interface in the lungs following inhalation.

Pulmonary Active Drugs

The pulmonary active drug portion of the conjugate may be any drug that has utility in the management or treatment of lung disease and, preferably are those characterized by a molecular weight below about 10,000 KD, e.g., a drug moiety having a molecular weight below about 5,000 KD, or about 2000 KD.

Pulmonary active drugs may include, but are not limited to elastase inhibitors, corticosteroids, bronchodilators, antihistamines, antibiotics, P2X and P2Y agonists, chemotherapeutic agents, matrix metalloproteinase inhibitors, ADAM inhibitors, kinase inhibitors, endothelin receptor antagonists, prostacyclin analogues, and phosphodiesterase type 5 inhibitors.

Exemplary elastase inhibitors include the compounds shown in FIG. 17. Other exemplary elastase inhibitors include the compounds described by Philip D. Edwards and Peter R. Bernstein in “Synthetic Inhibitors of Elastase,” Medicinal Research Reviews, Vol. 14, No. 2, 127-194 (1994), which is incorporated herein by reference. Two or more elastase inhibitors may be linked to a surface active agent of the invention and administered in combination. Alternatively, an elastase inhibitor may be linked to a surface active agent and administered in combination with a second elastase inhibitor linked to a surface active agent.

Exemplary corticosteroids that may be delivered to the lung include, but are not limited to, alclometasone, aldosterone, amcinonide, beclometasone, betamethasone, budesonide, ciclesonide, clobetasol, clobetasone, clocortolone, cloprednol, cortisone, cortivazol, deflazacort, deoxycorticosterone, desonide, desoximetasone, desoxycortone, dexamethasone, diflorasone, diflucortolone, difluprednate, fluclorolone, fludrocortisone, fludroxycortide, flumetasone, flunisolide, fluocinolone acetonide, fluocinonide, fluocortin, fluocortolone, fluorometholone, fluperolone, fluprednidene, fluticasone, fluticasone propionate, formocortal, halcinonide, halometasone, hydrocortisone/cortisol, hydrocortisone aceponate, hydrocortisone buteprate, hydrocortisone butyrate, loteprednol, medrysone, meprednisone, methylprednisolone, methylprednisolone aceponate, mometasone furoate, paramethasone, prednicarbate, prednisone, prednisolone, prednylidene, rimexolone, tixocortol, triamcinolone, and ulobetasol. Two or more corticosteroids may be linked to a surface active agent of the invention and administered in combination. Alternatively, a corticosteroid may be linked to a surface active agent and administered in combination with a second corticosteroid linked to a surface active agent.

Pulmonary active drugs may also include bronchodilators such as short-acting β2-adrenergicreceptor agonists, long-acting β2-adrenergic receptor agonists, short-acting anti-cholinergics, and long-acting anti-cholinergics. Non-limiting short-acting β2-adrenergic receptor agonists include salbutamol or albuterol, terbutaline, fenoterol, fenoterol hydrobromide, rimiterol, reproterol, pirbuterol, isoprenaline, orciprenaline, bitolterol, and broxaterol. Non-limiting long-acting β2-adrenergic receptor agonists include salmeterol, salmeterol xinafoate, formoterol, formoterol fumarate, clenbuterol, and procaterol. Non-limiting short-acting anti-cholinergics include ipratropium, ipratropium bromide, oxitropium and its salts. Non-limiting long-acting anti-cholinergics include tiotropium and tiotropium bromide monohydrate. Other bronchodialators may include, but are not limited to, aminophyliline, iorciprenaline, oxtriphylline, terbutaline sulfate, and theophylline. Two or more bronchodilators may be linked to a surface active agent of the invention and administered in combination. Alternatively, a bronchodilator may be linked to a surface active agent and administered in combination with a second bronchodilator linked to a surface active agent.

Pulmonary active drugs may also include antihistamines. Exemplary antihistamines include, but are not limited to as acrivastine, astemizole, azatadine, azelastine, brompheniramine, cetirizine, chlorpheniramine, cyclizine, cyproheptadine, diphehydramine, desloratidine, fexofenadine, hydroxyzine, levocetirizine, loratidine, meclizine, mepyramine, methscopolamine, misolastine, phenidamine, promethazine, pyrilamine, tecastemizole, and terfenadine. Two or more antihistamines may be linked to a surface active agent of the invention and administered in combination. Alternatively, an antihistamine may be linked to a surface active agent and administered in combination with a second antihistamine linked to a surface active agent.

U.S. Pat. No. 7,173,102, the disclosure of which is incorporated herein by reference, discloses anti-microbial polymers that are designed to possess amphiphilic properties in the presence of microbial cell walls and to disrupt the membrane and kill the organism. The polymeric materials are further designed to have low toxicity to mammalian cells. These facially amphiphilic polymers are capable of adopting repeating secondary structural motifs that allow for the segregation of polar and nonpolar regions of the molecule into different spatial regions. The anti-microbial polymers adopt amphiphilic conformations when placed in contact with the cell walls of microorganisms and the amphiphilic molecules are capable of disrupting essential cell wall functions resulting in the death of the microorganism with a much smaller death rate or no death rate for animal cells, thus providing a useful therapeutic margin for the treatment of infections. Many of these amphiphilic polymers are beta peptides. They can be synthesized as a leader or trailer sequence to the full length or a portion of a mammalian lung surfactant polypeptide or a mimic thereof to produce a potent lung targeted antimicrobial inhalant for the treatment of microbial lung infections including MRSA infections and various dangerous nosocomial infections.

Exemplary antibiotics that may be delivered to the lung include, but are not limited to penicillins, penicillins and beta-lactamase inhibitors, cephalosporins (generation I, II, III, and IV), macrolides and lincosamines, quinolones and fluoroquinoloes, carbepems, monbactams, aminoglycosides, glycopeptides, tetracylines, sulfonamides, rifampin, oxazolidones, streptogramins, sulfanomides, and others. Two or more antibiotics may be linked to a surface active agent of the invention and administered in combination. Alternatively, an antibiotic may be linked to a surface active agent and administered in combination with a second antibiotic linked to a surface active agent.

Exemplary penicillins include, but are not limited to, amoxicillin, ampicillin, bacampicillin, carbenicillin, carbenicillin indanyl, mezlocilin, piperacillin, and ticarcilin.

Exemplary penicillins and beta-lactamase inhibitors include, but are not limited to, amoxicillin-clavulanic acid, ampicillin-sulbactam, benzylpenicillin, cloxacilin, dicloxacilin, phenoxymethylpenicillin, carbenicillin, methicillin, oxacilin, penicillin G (benzathine, potassium, procaine), penicilin V, propicillin, epicillin, cyclacillin, piperacilin plus tazobactam, ticarcilllin plus clavulanic acid, and naficillin.

Exemplary cephalosporins (generation I) include, but are not limited to, cefadroxil, cefazolin, cephalexin, cephalothin, cephapirin, and cephradine. Cephalosporins (generation II) include, but are not limited to, cefaclor, cefamandole, cefoicid, ceforanide, cefoxitin, cefprozil, ceftmetazole, cefuroxime, cefuoxime axetil, and loracarbef. Cephalosporins (generation III) include, but are not limited to, cefdinir, ceftibuten, cefditoren, cefatamet, cefoperazone, cefixime, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, and ceftriaxone. Cephalosporins (generation IV) include, but are not limited to, cefepime.

Exemplary marcolides and lincosamine include, but are not limited to, azithromycin, clarithromycin, clindamycin, dirithromycin, erythromycin, lincomycin, telithromycine, and troleandomycin.

Monobactams include, but are not limited to, include aztreonam. Carbepenems include, but are not limited to, doripenem, imipenem-cilastatin, and meropenem.

Aminoglycosides include, but are not limited to, amikacin, amikacin sulfate, gentamicin, genatmicin sulfate, kanamycin, metilmicin, neomycin, netilmicin, streptomycin, tobramycin, and paromycin.

Glycopeptides include, but are not limited to, dalbavancin, oritavancin, telavancin, teicoplanin, and vancomycin.

Tetracylines include, but are not limited to, demclocylline, doxycycline, methacyline, minocyline, oxytetracycline, tetracyline, and chloretracycline.

Oxazolidonones include, but are not limited to linezolid. Streptogramins include but are not limited to quinoprisitin plus dalfopristin.

Sulfonamides include, but are not limited to mafenide, silver sulfadiazine, sulfacetamide, sulfadiazine, sulfamethoxazole, sulfasalzine, sulfanilamide, sulfisoxazole, trimethoprim-sulfamethoxazole, and sulfamethizole.

Other antibiotics include, but are not limited to, bacitracin, chloramphenical, Colistemetate, Fosfomycin, Isoniazid, Methenamine, Metronidazol, Mupirocin, Nitrofurantoin, Nitrofurazone, Novobiocin, Polymyxin (for example, Polymyxin B), Spectinomycin, Trimethoprim, Colistin, Cycloserine, Capreomycin, Pyrazinamide, Para-aminosalicyclic acid, Erythromycin ethylsuccinate plus sulfisoxazole, and tigecycline.

P2X agonists that may be delivered to the lung include, but are not limited to, ATP, and an ATP analogue (e.g., benzoyl derivatives of ATP). In one embodiment, ATP or an ATP analogue may be linked to a lung surfactant peptide for treatment as a tuberculosis-treating antibiotic. This will persist in the lung, bind to a P2X receptor expressed by macrophages harboring TB, and drive the macrophages along an apoptotic pathway, thus treating the latent TB. For disclosure of established techniques for chemically linking ATP or its analogs peptides, see, for example, Ahn, et al., “ATP-Conjugated Peptide Inhibitors for Calmodulin-Dependent Protein Kinase II,” Bioorganic and Medicinal Chemistry Letters, 2007. Two or more P2X agonists may be linked to a surface active agent of the invention and administered in combination. Alternatively, a P2X agonist may be linked to a surface active agent and administered in combination with a second P2X agonist linked to a surface active agent.

P2Y agonists that may be delivered to the lung include, but are not limited to denufosol tetrasodium, ATP, and an ATP analogue. P2Y agonists may be linked to a lung surfactant peptide for the treatment of cystic fibrosis. Two or more P2Y agonists may be linked to a surface active agent of the invention and administered in combination. Alternatively, a P2Y agonist may be linked to a surface active agent and administered in combination with a second P2Y agonist linked to a surface active agent.

Chemotherapeutic drugs that may delivered to the lung include, but are not limited to, alkylating agents, antiestrogens, aclarubicin, actinomycin D, aldesleukin, alemtuzumab, alitertinoin, allopurinol, altretamine, amifostine, anastrozole, asparaginase, bexarotene, bisantrene, bleomycin, busulfan, BCNU (carmustine), calusterone, capecitabine, carboplatin, celecoxib, chlorambucil, cisplatin, cladribine, cyclophosphamide, cyclooxygenase-2 inhibitor, cytarabine, CCNU (lomustine), dacarbazine, daunorubine, daunomycin, denileukin diftitox, dexrazoxane, diaziquone, docetaxel, doxorubicin, epirubicin, epoetin alfa, esorubicin. estramustine, etoposide (VP-16), exemestane, Filgrastim, floxuridine, fludarabine, 5-fluorouracil, fulvestrant, galactitol, gemcitabine, gemtuzumab, goserelin acetate, hydroxyurea, ibritumomab tiuxetan, idarubicin, ifosfamide, imatinib mesylate, interferon alpha, interferon gamma, iriniotecan, iroplatin, letrozole, leucovorin, levamisole, lonidamine, megrestrol acetate, melphalan, mercaptopurine, mesna, methotrexate, methoxsalen, mitomycin C, mitotane, mitoxantrone, mitoguazone, nandrolone phenpropionate, Nofetumomab, nitrogen mustard, oprelvekin, oxaliplatin, paclitaxel, pamidronate, pegademase, pegaspargase, pegfilgrastim, pentostatin, pipobroman, plicamycin, porfimer sodium, procarbazine, progestins, prednimustine, PCNU, quinacrine, rasburicase, rituximab, sargramostim, streptozocin, talc, tamoxifen, temozolomide, teniposide (VM-26), testolactone, thioguanine, thiotepa, topotecan, toremifene, tositumomab, trastuzumab, tertinoin, uracil mustard, valrubicin, vinblastine, vincristine, vindesine, vinorelbine, and zoledronate.

Two or more chemotherapeutic agents may be linked to a surface active agent of the invention and administered in combination. Alternatively, a chemotherapeutic agent may be linked to a surface active agent and administered in combination with a second chemotherapeutic agent linked to a surface active agent. Exemplary combination therapies include paclitaxel and carboplatin, cisplatin and vinorelbline tartrate, cisplatin and etoposide, and carboplatin and etoposide.

Pulmonary active drugs may include endothelin receptor antagonists. Exemplary endothelin receptor antagonists include, but are not limited to, tezosentan and bosentan, which are dual receptor antagonists affecting both endothelin A and endothlin B receptors, and sitaxentan, ambrisentan, and atrasentan, which affect endothelin A receptors.

Exemplary prostacylin analogues include, but are not limited to, Beraprost, Epoprostenol, Iloprost, and Treprostinil.

Exemplary phosphodiesterase type 5 inhibitors include, but are not limited to, sildenafil, tadalafil, and vardenafil.

Salts or solvates of the above identified pulmonary active drugs for use in the conjugates described herein are also contemplated.

Other exemplary inhibitors that may be linked to a surface active agent of the invention include, but are not limited to marimastat (a nonselective inhibitor of sheddase and matrix metalloproteinase enzymes), selective ADAM inhibitors such as INCB3619 (see Zhou et al., Cancer Cell, 10:39-50 (2006)), and EGFR tyrosine kinase inhibitors such as gefitinib and erlotinib. Two or more matrix metalloproteinase or kinase inhibitors may be linked to a surface active agent of the invention and administered in combination. Alternatively, a matrix metalloproteinase inhibitor (e.g., marimastat) may be linked to a surface active agent and administered in combination with a second agent (e.g., a second matrix metalloproteinase inhibitor, a kinase inhibitor, or a chemotherapeutic agent) linked to a surface active agent.

In other embodiments, the pulmonary active drug may be a polypeptide such as an antibody (e.g., an intact antibody, an antigen binding fragment such as a Fab, Fab′, (Fab′)₂, Fv fragment, or a biosynthetic antibody such as a single Fv or SFv molecule), a fusion protein, or a peptidomimetic, that binds an extracellular or cell surface target, e.g., an elastase, a TNF receptor, an EGF receptor, an adrenergic receptor, a P2X or P2Y purinergic receptor, or an endothelin receptor, or other target accessible to the pulmonary/gas interface. It is further contemplated that such polypeptide drugs may be directed against any target which is addressed by the drugs disclosed herein.

Linkage

Many strategies can be employed to link a pulmonary active drug to a surface active agent for use in the invention. Preferably, the pulmonary active drug can be attached to the surface active agent covalently either directly or using a linker that preserves the biological activity of the drug and retains significant dwell time of the surface active agent at the lung/air interface. In this case, at least one additional residue can be added at the amino- or carboxy-terminus or at an internal amino acid residue of a surfactant polypeptide of the type disclosed herein to generate a linker for covalently bonding a drug molecule. In an exemplary embodiment, SP-A, SP-B, SP-C, SP-D, or portions thereof, may be extended by at least one amino acid to create an unnatural amino acid or short amino acid sequence, e.g., four to eight amino acids long, by automated peptide synthesis. Alternatively, the native sequence of the human or animal form of these protein domains beyond these regions displaying the surfactant activity may be included as a natural linker.

A drug can be conjugated to the C-terminal or N-terminal amino acid of the surface active agent by bonding with the carboxyl group of the C-terminal amino acid or the amino group of the N-terminal amino acid. The drug may be bonded directly to the amino acid or via a linker. Representative covalent linkages include an ester, an amide, urea, carbamate, sulfonamide, ether, thioether, disubstituted amino, or a trisubstituted amine. (March, Advanced Organic Chemistry, 4th Ed., John Wiley & Sons, 1992.) Other linkage types could also be used. One strategy is to synthesize a derivative of the pulmonary active drug as may be necessary in specific cases to create a selectively reactive chemical group in a region of the molecule chemically separate from its active region at locations suggested by structure function analysis studies.

One type of covalent linker comprises amino acid residues. Such linkers may comprise at least one residue or can be 40 or more residues, more often 1 to 10 residues, and most often 1 to 5 or 5-10 amino acid residues in length. The linker is usually a small, water-soluble, neutral polar or non-polar amino acid or unstructured peptide. Typical amino acid residues used for linking are glycine, tyrosine, cysteine, lysine, glutamic acid, and aspartic acid, or the like. One linker frequently used where linked moieties each are intended to retain their independent function is a glycine rich sequence comprising between one to five glycine residues. Another linker frequently used in similar contexts where linked moieties each are intended to retain their independent function is Glycine and Serine rich synthetic sequences such as Gly Gly Ser Gly Gly Ser Gly Gly Gly Gly Ser Gly Ser (SEQ ID NO: 95), or [Gly Gly Gly Gly Ser]_(n)(SEQ ID NO: 96) where n is one, two, or three.

In another embodiment, a pulmonary active drug may can be chemically linked to a surfactant moiety through a succinic acid-peptide linker. For example, a steroid, such as prednisone, can be bonded to succinic acid to form a steroid-succinate (e.g., a prednisone-succinate) then conjugated to a peptide linker, such as a glycine linker (e.g., [Gly]_(n), where n is 1-5), as described by Penugonda et al. (J. Pharm. Sci. 2008, 97(7):2649-2664), which is incorporated herein by reference. The steroid-succinate-peptide conjugate can then be further conjugated with a surfactant moiety through the carboxyl group on the terminus of the glycine linker.

Other strategies for attaching a pulmonary active agent to a surface active agent are contemplated including by chelation, ionic attraction, or non-covalent association such as hydrophobic-hydrophobic interaction.

The chemical structure of the pulmonary active drug may also guide the site of attachment to a surfactant moiety. For example, many antihistamines have a biphenyl-like aromatic core with a basic nitrogen spaced about four atoms from the center of the aromatic core. Early generation antihistamines have short side chains, whereas newer generation antihistamines have longer side chains extending from the aromatic core. FIG. 20 is an exemplary collection of antihistamines, which demonstrates that large variations are well-tolerated on the antihistamine side chains, both short and long. As such, it is contemplated that attaching the surfactant moiety to the side chain of the antihistamine should not significantly impact the therapeutic properties of the antihistamine. For example, it is contemplated that attaching a surfactant moiety to the side chain hydroxyl group in hydroxyzine or terfenadine using a succinate-peptide linker will not significantly impact the therapeutic properties of these antihistamines. Similarly, it is contemplated that a surfactant moiety may be attached to the terminal carboxyl group of certirizine either directly or using a glycine linker. Further, it is contemplated that a surfactant moiety may be attached to the nitrogen atom of the terminal amide group of marimastat using a glycine linker as illustrated in FIG. 21.

Representative synthetic procedures for attaching various pulmonary active drugs to a polypeptide surfactant moiety either directly or using a linker are shown below in Scheme A.

Further, one of skill in the art will appreciate, based on the teachings herein, that minor structure changes can be made to various pulmonary active drugs to make them more suitable for attachment to the surfactant moiety. For example, it is contemplated that one of the N-methyl groups of diphenhydramine could be replaced with a hydroxy ethylene group suitable for attachment to the surfactant moiety. Similarly, it is contemplated that one of the methoxy group of astemizole can be replaced with a hydroxyl group suitable for attachment to the surfactant moiety.

Once a pulmonary active drug and surfactant moiety have been selected, a library of fusion constructs may then be created, comprising individual species exploiting different points of attachment on the chemical structure of the drug and on the peptide, different length linkers, different linker chemistries, different length surfactant peptides, etc., all with a view to improving the binding constant of the drug to its target, improving activity, reducing immunogenicity, or for other purposes. The desired combination of different surfactant polypeptide domains, linkers, and attachment points can be generated, for example, by brute force construction of a desired number of candidate constructs. A library of such constructs may be generated using standard molecular biology protocols. As noted, the drug may be attached at either the N-terminal or the C-terminal, or at an intermediate location. The size/length, and amino acid sequence of the mammalian lung surfactant polypeptide or a mimic thereof may be varied. Nucleic acids encoding the various mammalian lung surfactant polypeptide or a mimic thereof can be recombinantly fused and cloned in suitable expression vectors, under the control of operatively linked promoters and transcription regulators. The construct may also be post translationally modified as may be necessary or desirable in specific instances, e.g., glycosylated or pegylated.

The resulting library can be screened for the ability to modify the specific target of interest in the lungs of experimental animals. An assay for the appropriate biological function can be used to quantitate the potency of the drug in vivo. For example, the assay to screen the library of fusion constructs can be one which measures the biological activity of the target molecule or a downstream molecule that is regulated by the target molecule such as a protocol described in the examples disclosed below.

Once one or a group of surfactant active agents are optimized for safety, distribution and dwell time within the lungs, by appropriate screening or otherwise, its properties may be further enhanced by one or more rounds of mutagenesis and additional selection/screening according to art known methods. This will lead to a functionally optimized structure, which can be used repeatedly to enhance the efficacy within the lungs of a wide variety of different pulmonary active drug classes and individual species for the treatment of various lung diseases.

Methods of Administration

Compositions of the invention are delivered to the lungs by inhalation. Inhalation devices, such as inhalers (including dry powder inhaler and metered dose inhalers) and nebulizers (also known as atomizers) may be used to deliver the disclosed compositions to the lungs. Exemplary dry powder inhalers can be obtained from Inhale Therapeutic Systems as described in U.S. Pat. Nos. 5,458,135; 5,740,794; 5,785,049, which are herein incorporated by reference. Dry powder inhalers can also be obtained from 3M as described in U.S. Pat. No. 6,029,661, which is incorporated herein by reference.

The compositions disclosed herein may also be administered using a metered dose inhaler (MDI) containing a solution or suspension of drug in a pharmaceutically inert liquid propellant, e.g., a chlorofluorocarbon (CFC) or fluorocarbon, as described in U.S. Pat. Nos. 5,320,094 and 5,672,581, both incorporated herein by reference. Metered dose inhalers are designed to deliver a fixed unit dosage of medicament per actuation or “puff”, for example in the range of 10 to 5000 microgram medicament per puff. Exemplary metered dose inhibitors can be obtained from 3M as described in U.S. Pat. Nos. 5,224,183; 5,290,534; 5,511,540; 6,454,140; and 6,615,826, which are incorporated herein by reference. Metered dose inhalers may also be CFC-free. Drug compositions to be used with an inhaler may be in the form of aerosolized solid particles or droplets of liquid or suspension.

Alternatively, the compositions described herein may be dissolved or suspended in a solvent, e.g., water or saline, and administered by nebulization. Exemplary nebulizers for delivering an aerosolized solution include the AERx™ (Aradigm), the Ultravent®(Mallinkrodt), the Pari LC Plus™ or the Pari LC Star™ (Pari GmbH, Germany), the DeVilbiss Pulmo-Aide, and the Acorn II® (Marquest Medical Products).

Drug Formulation

Drug compositions disclosed herein can be formulated into a solution and/or a suspension of particles in a carrier appropriate for inhalation into the respiratory tract and the lungs. Such carriers are also well known to the ordinary artisan familiar with inhalants for the delivery of fine droplets and insufflations for the delivery of inhalable fine particles, on the order of, for example, from about 0.5 to 1 micron, and preferably from about 0.5 to about 0.7 micron, comprised of powders, mists or aerosols, into the respiratory tract as described in Remington's Pharmaceutical Sciences, 16th edition, 1980, Ed. By Arthur Osol, which is incorporated herein by reference.

In one embodiment, drug compositions for inhalation administration can be administered as powders. The powdered drug or composition is normally located within a container such as a hard gelatin capsule or a blister package, or a multi-dose devise. The capsule or blister is ruptured or broached within in an inhaler device, thereby enabling the powder to be inhaled. Generally, the mean particle size of the drug used for inhalation is between 1 and 10 micron with the size range between 2 and 5 microns being particularly suitable for penetrating the peripheral airways of the lungs. Such particle size ranges are commonly achieved by micronisation or spray drying.

A powdered drug composition is often administered as a composition comprising a blend or mixture of the medicament with an inert carrier. Usually the inert carrier has a mean particle size substantially larger than that of the drug. This provides, among other advantages, an improvement in the flow properties and dispensing accuracy of the composition.

Commonly described carrier materials for produced drug, include calcium carbonate and sugars, for example sucrose, mannitol or dextrose or, more particularly, lactose, which are pharmaceutically acceptable and pose no problems of toxicity, since any residues imbibed during dosing are well tolerated upon digestion or may be easily eliminated by disillusion (e.g., in the case of the sugars) or mucocilliary clearance from the lung.

The composition in the capsule or blister is frequently about 25 mgs. This weight probably represents the maximum quantity of powder that may be comfortably inhaled without undue side effects, such as coughing, and also corresponds to the minimum quantity that is usually dispensed by filling machines.

In certain embodiments, compositions formulated for powder inhalation comprise a carrier present at a concentration of about 95.0 to 99.99%. More particularly, from 97.0 to 99.9%, especially from 98.0 to 99.8%, by weight. Processes for preparing such powders, by the application or adaptation of known methods, also constitute features of the invention.

In other embodiments, the drug composition may be formulated as an aerosol formulation using methods well known in the art. One widely used method for dispensing such an aerosol formulation involves making a suspension formulation of the drug as a finely divided powder in a liquefied propellant gas. Alternatively a solution formulation can be prepared where the drug is dissolved in a propellant system, perhaps containing solubilizers and co-solvents to aid dissolution of the drug. Pressurized metered dose inhalers (pMDI) are normally used to dispense such formulations to a patient. Propellants may include chlorofluorocarbon (CFC), fluorocarban (FC), or hydrofluoroalkane (HFA) propellants.

Methods of Treatment

The disclosure also provides methods for treating a subject suffering from lung inflammation or lung disease. In another embodiment, the disclosure provides a method for treating a subject who is at risk of suffering from a lung disorder. The method comprises administering to the subject a conjugate comprising a pulmonary active drug covalently bonded to a surface active agent, which has an affinity for the human alveolar/gas interface and which comprises at least a portion of a mammalian lung surfactant polypeptide or a mimic thereof that is substantially non-immunogenic to humans. The conjugate is administered to the subject by inhalation in an amount effective to induce a drug effect in the lungs. The subject may be a human, monkey, chimpanzee, horse, dog, cat, cow, sheep, pig, rat or mouse. In exemplary embodiments, the subject is a human.

The subject in need of treatment is suffering from lung inflammation or is suffering from or at risk of suffering from lung disease. Exemplary lung diseases that may be treated with the drug composition described herein include, but are not limited to, emphysema, chronic bronchitis and acute exacerbation of chronic bronchitis (AECB), chronic obstructive pulmonary disease (COPD), asthma, respiratory distress disorder (RDS), pneumonia (including ventilator associated pneumonia), tuberculosis or other bacterial infection, cystic fibrosis, pulmonary arterial hypertension, and/or lung cancer.

Dosage

The administration of pulmonary active drugs conjugated to a surface active agent reduces the dosing frequency relative to administration of an unconjugated drug. In certain embodiments, the administration step may be repeated once daily, every other day, every three days, every four days, every five days, biweekly, or weekly.

EXAMPLES

The invention is further illustrated by the following examples. The examples are provided for illustrative purposes only, and are not to be construed as limiting the scope or content of the invention in any way.

Example 1 Conjugation of Human Neutrophil Elastase Inhibitors to a SP-B peptide

A panel of potent small molecule human neutrophil elastase (HNE) inhibitors can be conjugated to a SP-B peptide comprising the amino terminal 25 amino acids of SP-B (FPIPLPYCWLCRALIKRIQAMIPKG) (SEQ ID NO: 88). The total molecular weight of the SP-B peptide comprising the amino terminal 25 amino acids is 2926.97 after water molecule elimination. The HNE inhibitors are conjugated to the SP-B N-terminal 25-mer using a glycine linker similar to the linkage depicted in FIG. 18.

Example 2 Synthesis of Target B

All solvents used for the reaction were LR grade solvents. Room temperature (RT) indicates temperature ranging from 27-32° C. All the reactions were monitored by TLC unless specified. Solutions were evaporated under reduced pressure using rotary evaporator. NMR was taken on Varian 400 MHz. Column chromatography was done using silica gel 100-200 mesh unless specified.

Synthesis of Stage 1

A solution of Cbz-Val-Pro-OH (5 g, 14 mmol) in dry tetrahydrofuran (85 mL) was cooled to −20° C. under nitrogen. N-methylmorpholine (1.74 mL, 15 mmol) followed by isobutyl chloroformate (2 mL, 15 mmol) was added to the reaction mixture. The reaction mixture was stirred at −20° C. for 15 min and then cooled to −40° C. A solution of L-Valinol (1.62 g, 15 mmol) in tetrahydrofuran (25 mL) was added drop wise to the reaction mixture. The reaction mixture was allowed to warm to room temperature overnight. The reaction mixture was filtered and the filtrate was diluted with ethyl acetate (60 mL). The combined organic layers were washed successively with 1N HCl (60 mL), NaHCO₃ (30 mL) and brine (30 mL). The organic layer was dried over sodium sulphate, filtered and concentrated to give the desired product (5.7 g). HPLC Rt: 5.76; LCMS (M+1): 434; Yield: 92.5%.

Synthesis of Stage 2

A solution of oxalyl chloride (2.45 mL, 28 mmol) in dry dichloromethane (110 mL) was cooled to −60° C. and a solution of DMSO (4.09 mL, 57.7 mmol) in dichloromethane (35 mL) was added drop wise over a period of 1 h, maintaining the reaction mixture temperature at −45° C. The reaction mixture was allowed to warm to −30° C. and a solution of stage-1 (6.1 g, 14 mmol) in dichloromethane (35 mL) was added drop wise over a period of 1 h. The reaction mixture was stirred at −25° C. for 1 h. The reaction mixture was cooled to −40° C. and diisopropylethylamine (10 mL, 57.7 mmol) was added drop wise over a period of 1 h. The reaction was warmed to room temperature and then washed with 1 N HCl (60 mL) and brine (60 mL). The organic layer was dried over sodium sulphate, filtered and the filtrate was concentrated under reduced pressure to give the desired product (5.7 g). HPLC Rt: 6.81; LCMS (M+1): 432; Yield: 94%.

Synthesis of Stage 3

A suspension of Zinc (2.5 g, 39 mmol) and stage-2 (5.64 g, 13 mmol) in dry tetrahydrofuran (100 mL) was heated to 60° C. under nitrogen atmosphere. Ethyl bromodifluoroacetate (7.93 g, 39 mmol) was added and the reaction was heated to 60° C. for 1 h. The reaction mixture was cooled to room temperature, tetrahydrofuran was removed under reduced pressure and ethyl acetate (100 mL) was added. The reaction mixture was washed with 1M KHSO₄ (50 mL) and brine (50 mL) and dried over sodium sulphate. The organic layer was filtered and concentrated under reduced pressure to give the crude product, which was then purified by preparative HPLC to give the desired product (2.9 g). HPLC Rt: 7.44; LCMS (M+1): 556; Yield: 40%.

Synthesis of Stage 4

A solution of stage-3 (1.8 g, 3.2 mmol) and benzyl amine (1.06 mL, 9.7 mmol) in ethanol (40 mL) was stirred at reflux for 4 h. The reaction mixture was cooled to room temperature and stirred for an additional 48 h under nitrogen atmosphere. The solvent was removed under reduced pressure. The residue was dissolved in ethyl acetate (50 mL) and the solution was washed with 1N HCl (20 mL) and brine (20 mL). The organic layer was dried over sodium sulphate, filtered and the solvent was removed under vacuum to give the desired product (1.7 g). HPLC Rt: 7.56; LCMS (M+1): 616; Yield: 88%.

Stage-5

A mixture of stage-4 (1.85 g, 3 mmol) and 20% palladium hydroxide (1 g) in ethyl acetate (50 mL) was placed in a pressure vessel at 200 psi for 20 h. The reaction mixture was filtered through celite and the filtrate was concentrated under vacuum to give the desired product along with some starting material, which was used as such for further reaction without any purification (1.1 g). HPLC Rt: 4.90; LCMS (M+1): 482; Yield: 80%.

Stage-6

To a solution of stage-5 (1 g, 2.1 mmol) in dry dichloromethane (50 mL) at 0° C. was added triethylamine (0.25 mL, 1.8 mmol). Then a solution of Ethyl oxalyl chloride (0.2 mL, 1.87 mmol) in dichloromethane (20 mL) was added drop wise. The reaction mixture was allowed to warm to room temperature and stirred for further 2 h. The reaction mixture was quenched with water (25 mL). The organic layer was separated, dried over sodium sulphate and evaporated under reduced pressure to give the desired product, which was then purified by preparative HPLC. HPLC Rt: 6.72; LCMS (M+1): 583; Yield: 0.5 g (42%).

Stage-7

Trifluoroacetic acid (0.92 mL, 12.02 mmol) was added to a stirred solution of stage-6 (1.75 g, 3 mmol) and Dess-Martin periodinane (5.1 g, 12.02 mmol) in dry dichloromethane (25 mL). The mixture was stirred for 4 h at room temperature under nitrogen atmosphere. Ethyl acetate (100 mL) was added and the mixture was washed with saturated sodium thiosulphate (60 mL), saturated NaHCO₃ (60 mL) and brine (60 mL). The organic layer was dried over sodium sulphate, filtered and evaporated under reduced pressure to give the desired product, which was used as such for the further step. HPLC Rt: 7.32; LCMS (M+1): 581; Yield: 1.7 g (97%).

Stage-8

A solution of stage-7 (2.2 g, 3.7 mmol) in methanol water 1:1 (20 mL) was treated with 1N NaOH (0.18 g, 4.5 mmol) and stirred at room temperature for 4 h. The reaction mixture was concentrated to remove methanol, acidified with 1N HCl and extracted with ethyl acetate (60 mL). The organic layer was dried over sodium sulphate and evaporated under reduced pressure to give the desired product, which was then purified by preparative HPLC. HPLC Rt: 6.47-6.61; LCMS (M+1): 553; Yield: 500 mg (24% After prep).

Stage-9

Wang resin (2.5 g, 0.5 eq) was treated with 20% piperidine in DMF (15 mL) and stirred for about an hour. It was then washed with DMF (2 times, 15 mL), DCM (3 times, 15 mL) and then dried under vacuum. Kaiser test was performed on the resin to ensure complete removal of F-moc group. Then it was used for coupling with the stage −8 product. To a solution of the resin in DMF was added a solution of the above acid (97 mg, 0.176 mmol) in DMF (5 mL), followed by the addition of PyBoP (91 mg, 0.176 mmol) and N-methyl morpholine (0.24 mL, 0.22 mmol). The reaction mixture was allowed to shake on a shaker for about 3 h. The solution was decanted, fresh lot of the above reagents was again added and it was further allowed to shake for 3 h. This process was repeated for about 4 times. The solution was decanted, and the resin was washed with DMF (3 times, 15 mL), DCM (3 times, 15 mL) dried under vacuum. Kaiser test was performed on the resin to ensure that the coupling has taken place.

Stage-10

Procedure for making the cleavage solution. TFA (81%), Phenol (5%), Thioanisole (5%), 1,2,Ethanedithiol (2.5%), Water (3%), Dimethylsulphide (2%), ammonium iodide (1.5%). To the resin was added the cleavage solution and was allowed to shake on a shaker for about 3 h. The resin was filtered through cotton and washed with TFA. The filtrate was then concentrated under vacuum and triturated with cold ether to give a white solid, which was then purified by preparative HPLC to give the desired product. LCMS (M/3): 1155; Yield: 46 mg (1.84%).

Example 3 Synthesis of Target C Stage-6

To a solution of stage-5 (3.2 g, 6.6 mmol) in dry THF (50 mL) at room temperature was added mono ethyl malonate (0.7 mL, 6 mmol) followed by HOBT (1.63 g, 12 mmol), EDCI (1.27 g, 6.6 mmol) and N-methyl morpholine (1.6 mL, 15 mmol). The reaction mixture was then allowed to stir at room temperature for 4 h. The reaction mixture was then quenched with water and evaporated under reduced pressure in order to remove THF. The aqueous layer was then extracted with ethyl acetate (100 mL). The organic layer was dried over sodium sulphate and concentrated to give the crude product which was then purified by preparative HPLC (0.98 g). HPLC Rt: 6.44; LCMS (M+1): 596; Yield: 25% After prep.

Stage-7

Trifluoroacetic acid (0.45 mL, 5.7 mmol) was added to a stirred solution of stage-6 (0.86 g, 1.45 mmol) and Dess-Martin periodinane (2.46 g, 5.7 mmol) in dry dichloromethane (30 mL). The mixture was stirred for 4 h at room temperature under nitrogen atmosphere. Ethyl acetate (50 mL) was added and the mixture was washed with saturated sodium thiosulphate (20 mL), saturated NaHCO₃ (20 mL) and brine (20 mL). The organic layer was dried over sodium sulphate, filtered, and the solvent was removed under vacuum to give the desired product (0.74 g). HPLC Rt: 7.05; LCMS (M+1): 595; Yield: 86%.

Stage-8

A solution of stage-7 (0.75 g, 1.2 mmol) in methanol water 1:1 (5 mL:5 mL) was treated with 1N NaOH (0.060 g, 1.5 mmol) and stirred at room temperature for 4 h. The reaction mixture was concentrated to remove methanol, acidified with 1N HCl (till pH 2) and extracted with ethyl acetate (50 mL). The organic layer was dried over sodium sulphate and concentrated under reduced pressure to give the desired product, which was then purified by preparative HPLC (0.4 g). HPLC Rt: 6.01-6.51; LCMS (M+1): 567; Yield: 56% After prep.

Stage-9

Wang resin (2 g, 0.08 mmol) was treated with 20% piperidine in DMF (20 mL) and stirred for about an hour. It was then washed with DMF (2 times), DCM (3 times) and then dried under reduced pressure. Kaiser test was performed on the resin to ensure complete removal of F-moc group. Then it was used for coupling with the stage −8. To a solution of the resin (2 g, 0.08 mmol) in DMF (5 mL) was added a solution of the above acid (0.1 g, 0.16 mmol) in DMF (3 mL), followed by the addition of PyBoP(0.83 g, 0.16 mmol) and N-methyl morpholine (0.2 mL, 0.2 mmol). The reaction mixture was allowed to shake on a shaker for about 3 h. The solution was decanted, fresh lots of the above reagents were again added and it was further allowed to shake for 3 h. This process was repeated for about 4 times. The solution was decanted, and the resin was washed with DMF (3 times), DCM (3 times) dried under reduced pressure. Kaiser test was performed on the resin to ensure that the coupling has taken place.

Stage-10

Procedure for making the cleavage solution. TFA (81%), Phenol (5%), Thioanisole (5%), 1,2,Ethanedithiol (2.5%), Water (3%), Dimethylsulphide (2%), ammonium iodide (1.5%). To the resin was added the cleavage solution (25 mL) and was allowed to shake on a shaker for about 3 h. The resin was filtered through cotton and washed with TFA. The filtrate was then concentrated under reduced pressure and triturated with cold ether to give a white solid, which was then purified by preparative HPLC to give the desired product (0.080 g). LCMS (M/3): 1160; Yield: 2.4%.

Example 4 Bronchoaveolar Lavage of the Lungs following HNE Exposure

A total of 14 animals were used in this study. Anesthetized mice received 0.1 ml of solution delivered by a blunt tip catheter to the opening of the trachea and the animals allowed to aspirate the solution into their lungs (this method is also referred to as intratracheal instillation). All animals were exposed to the treatment for 2 hours and re-anesthetized. Bronchoalveolar lavage of the lungs was performed as described below. The four animals given HNE (the causative agent of emphysema) had an average lung blood cell level by weight of 0.053 grams. Target 2 (a potent HNE inhibitor developed by Zeneca in the early 1990s) when given with the HNE, reduced the average blood level in the lungs by 34% to 0.035 grams. Target C (the Zeneca molecule covalently attached to the N-terminus of the first 25 residues of the human surfactant B peptide) when given with the HNE reduced the average blood level in the lungs by 87% to 0.007 grams.

A set of animals was used to determine the optimal concentration of HNE for the study. An initial sample exposed to 50 micrograms of HNE did not survive exposure. The amount of HNE was reduced to 40 micrograms to maintain a maximal detrimental but survivable effect. The animals exposed to 40 micrograms of HNE were able to survive long enough to perform the complete study. All animals remaining in the complete study were exposed to 40 micrograms of HNE.

Target 2 (the Zeneca molecule) was given in a 70-fold molar excess relative to HNE. In the kinetic studies, Target 2 has 2 nM affinity. Target C (the Zeneca molecule covalently attached to the first 25 residues of the human surfactant peptide B) loses approximately two orders of magnitude of potency as compared to Target 2 in the kinetic studies and thus Target C has approximately 200 nM affinity for HNE. Accordingly, the Target C was given to the animals in a 100 fold molar excess relative to HNE in these studies.

Bronchio-Alveolar lavage (BAL) is performed by 1) anesthetizing the animals; 2) performing a tracheotomy on the animals and cutting open the chest and spreading the ribs to enable full lung expansion; 3) pressurizing 1 ml of PBS buffer solution into the lungs through the tracheotomy catheter; 4) sucking the solution out of the lungs; 5) centrifuging the recovered solution to separate the red blood cell fraction from the liquid fraction; and 6) weighing the red blood cell fraction.

TABLE 2 Lung Blood Cell Level Measurements Mouse Mouse Mouse Mouse Group Treatment group 1 group 2 group 3 Group 4 mean PBS/DMSO 0 0 0 Not done 0 HNE/DMSO 0.09 g 0.049 g 0.021 g 0.051 g 0.053 g Target 2 + HNE 0.03 g 0.041 g  0.03 g 0.039 g 0.035 g Target C + HNE 0 0.007 g 0.014 g Not done 0.007 g

Example 5 Intratracheal Delivery of HNE, HNE mixed with Zeneca Inhibitor, and HNE mixed with Zeneca Inhibitor Linked to a Lung Surfactant Peptide

A total of 42 animals were used in this study. Anesthesized mice received 0.1 ml of solution delivered by intratracheal instillation as described above in Example 4. Animals were divided into the following treatment groups: four animals received saline only; ten animals received HNE only; four animals received the Zeneca inhibitor (target 2) only; eight animals received HNE mixed with the Zeneca inhibitor (target 2); eight animals received Zeneca inhibitor linked to a lung surfactant peptide (target C) only; and eight animals received HNE mixed with Zeneca inhibitor linked to a lung surfactant peptide (target C). Animals receiving HNE were instilled with 40 micrograms HNE. Target 2 (Zeneca inhibitor) was administered in a 70-fold molar excess relative to HNE and Target C (Zeneca inhibitor linked to a lung surfactant peptide) was administered in a 100 fold molar excess relative to HNE as described in Example 4.

All animals were observed a four week period prior to sacrifice following IRB approved protocols and the lungs from each animal were removed for sectioning. The Hilar and central region was sectioned from the left lung and the caudal region was sectioned from the right lung of each animal. Lung sections were scored using blind-scoring by an expert as “E” for emphysema and “N” for no emphysema.

Target 2 (the Zeneca inhibitor) had no effect on HNE induced emphysema (compare FIG. 23, which shows lung histology sections of mice instilled with HNE and Zeneca inhibitor, with FIG. 22, which shows lung histology sections of mice with HNE induced emphysema). In contrast, target C (the Zeneca inhibitor covalently attached to a lung surfactant peptide) strongly inhibited HNE induced emphysema (compare FIG. 25, which shows lung histology sections of mice instilled with HNE and Zeneca inhibitor linked to a lung surfactant peptide with FIG. 22). Further, lung histology sections from animals instilled with HNE and target C (Zeneca inhibitor linked to a lung surfactant peptide) could not be distinguished in blind scoring from lung histology sections from animals receiving saline only (compare FIG. 25 with FIG. 24).

The distance between adjacent alveolar walls was also measured to quantitate the degree of lung destruction for each treatment group. Lung sections obtained from each animal were analyzed under a microscope (which is connected to a computer) and lines were drawn over the lung sections between adjacent alveolar walls. The distance along the lines was calculated to determine the average distance or mean linear intercept (MLI) between adjacent alveolar walls. An increase in the average distance between adjacent alveolar walls corresponds to an increase in lung destruction.

As shown in Table 3 below, exposure with HNE increased the MLI compared to the negative control (saline only) treatment group. Target 2 had no effect on HNE induced emphysema (HNE+Target 2), whereas target C(HNE+Target C) strongly inhibited HNE induced emphysema (i.e., shows a decrease in MLI compared to HNE only exposure and returns to the saline only baseline). Target 2 only and Target C only are negative controls.

TABLE 3 Distance between Adjacent Alveolar Wall Measurements Treatment Group Animals (N) MLI (μm) Standard Deviation Saline only 4 44.40 6.50 HNE only 10 51.45 5.78 Target 2 only 4 43.27 3.82 HNE + Target 2 8 53.27 8.27 Target C only 8 40.94 1.59 HNE + Target C 8 45.82 3.30

Incorporation by Reference

The entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes.

Equivalents

The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

TABLE 1 Exemplary human SP-B peptides (C-terminal truncations) Human SP-B peptides SEQ ID NO FPIPLPYCWLCRALIKRIQAMIPKGALAVAVAQVCRVVPLVAGGICQCLA  SEQ ID NO: 36 ERYSVILLDTLLGRMLPQLVCRLVLRC FPIPLPYCWLCRALIKRIQAMIPKGALAVAVAQVCRVVPLVAGGICQCLA SEQ ID NO: 37 ERYSVILLDTLLGRMLPQLVCRLVLR FPIPLPYCWLCRALIKRIQAMIPKGALAVAVAQVCRVVPLVAGGICQCLA SEQ ID NO: 38 ERYSVILLDTLLGRMLPQLVCRLVL FPIPLPYCWLCRALIKRIQAMIPKGALAVAVAQVCRVVPLVAGGICQCLA  SEQ ID NO: 39 ERYSVILLDTLLGRMLPQLVCRLV FPIPLPYCWLCRALIKRIQAMIPKGALAVAVAQVCRVVPLVAGGICQCLA  SEQ ID NO: 40 ERYSVILLDTLLGRMLPQLVCRL FPIPLPYCWLCRALIKRIQAMIPKGALAVAVAQVCRVVPLVAGGICQCLA  SEQ ID NO: 41 ERYSVILLDTLLGRMLPQLVCR FPIPLPYCWLCRALIKRIQAMIPKGALAVAVAQVCRVVPLVAGGICQCLA  SEQ ID NO: 42 ERYSVILLDTLLGRMLPQLVC FPIPLPYCWLCRALIKRIQAMIPKGALAVAVAQVCRVVPLVAGGICQCLA  SEQ ID NO: 43 ERYSVILLDTLLGRMLPQLV FPIPLPYCWLCRALIKRIQAMIPKGALAVAVAQVCRVVPLVAGGICQCLA  SEQ ID NO: 44 ERYSVILLDTLLGRMLPQL FPIPLPYCWLCRALIKRIQAMIPKGALAVAVAQVCRVVPLVAGGICQCLA  SEQ ID NO: 45 ERYSVILLDTLLGRMLPQ FPIPLPYCWLCRALIKRIQAMIPKGALAVAVAQVCRVVPLVAGGICQCLA  SEQ ID NO: 46 ERYSVILLDTLLGRMLP FPIPLPYCWLCRALIKRIQAMIPKGALAVAVAQVCRVVPLVAGGICQCLA  SEQ ID NO: 47 ERYSVILLDTLLGRML FPIPLPYCWLCRALIKRIQAMIPKGALAVAVAQVCRVVPLVAGGICQCLA  SEQ ID NO: 48 ERYSVILLDTLLGRM FPIPLPYCWLCRALIKRIQAMIPKGALAVAVAQVCRVVPLVAGGICQCLA  SEQ ID NO: 49 ERYSVILLDTLLGR FPIPLPYCWLCRALIKRIQAMIPKGALAVAVAQVCRVVPLVAGGICQCLA  SEQ ID NO: 50 ERYSVILLDTLLG FPIPLPYCWLCRALIKRIQAMIPKGALAVAVAQVCRVVPLVAGGICQCLA  SEQ ID NO: 51 ERYSVILLDTLL FPIPLPYCWLCRALIKRIQAMIPKGALAVAVAQVCRVVPLVAGGICQCLA  SEQ ID NO: 52 ERYSVILLDTL FPIPLPYCWLCRALIKRIQAMIPKGALAVAVAQVCRVVPLVAGGICQCLA  SEQ ID NO: 53 ERYSVILLDT FPIPLPYCWLCRALIKRIQAMIPKGALAVAVAQVCRVVPLVAGGICQCLA  SEQ ID NO: 54 ERYSVILLD FPIPLPYCWLCRALIKRIQAMIPKGALAVAVAQVCRVVPLVAGGICQCLA  SEQ ID NO: 55 ERYSVILL FPIPLPYCWLCRALIKRIQAMIPKGALAVAVAQVCRVVPLVAGGICQCLA  SEQ ID NO: 56 ERYSVIL FPIPLPYCWLCRALIKRIQAMIPKGALAVAVAQVCRVVPLVAGGICQCLA  SEQ ID NO: 57 ERYSVI FPIPLPYCWLCRALIKRIQAMIPKGALAVAVAQVCRVVPLVAGGICQCLA  SEQ ID NO: 58 ERYSV FPIPLPYCWLCRALIKRIQAMIPKGALAVAVAQVCRVVPLVAGGICQCLA  SEQ ID NO: 59 ERYS FPIPLPYCWLCRALIKRIQAMIPKGALAVAVAQVCRVVPLVAGGICQCLA  SEQ ID NO: 60 ERY FPIPLPYCWLCRALIKRIQAMIPKGALAVAVAQVCRVVPLVAGGICQCLA  SEQ ID NO: 61 ER FPIPLPYCWLCRALIKRIQAMIPKGALAVAVAQVCRVVPLVAGGICQCLA  SEQ ID NO: 62 E FPIPLPYCWLCRALIKRIQAMIPKGALAVAVAQVCRVVPLVAGGICQCLA  SEQ ID NO: 63 FPIPLPYCWLCRALIKRIQAMIPKGALAVAVAQVCRVVPLVAGGICQCL SEQ ID NO: 64 FPIPLPYCWLCRALIKRIQAMIPKGALAVAVAQVCRVVPLVAGGICQC SEQ ID NO: 65 FPIPLPYCWLCRALIKRIQAMIPKGALAVAVAQVCRVVPLVAGGICQ SEQ ID NO: 66 FPIPLPYCWLCRALIKRIQAMIPKGALAVAVAQVCRVVPLVAGGIC SEQ ID NO: 67 FPIPLPYCWLCRALIKRIQAMIPKGALAVAVAQVCRVVPLVAGGI SEQ ID NO: 68 FPIPLPYCWLCRALIKRIQAMIPKGALAVAVAQVCRVVPLVAGG SEQ ID NO: 69 FPIPLPYCWLCRALIKRIQAMIPKGALAVAVAQVCRVVPLVAG SEQ ID NO: 70 FPIPLPYCWLCRALIKRIQAMIPKGALAVAVAQVCRVVPLVA SEQ ID NO: 71 FPIPLPYCWLCRALIKRIQAMIPKGALAVAVAQVCRVVPLV SEQ ID NO: 72 FPIPLPYCWLCRALIKRIQAMIPKGALAVAVAQVCRVVPL SEQ ID NO: 73 FPIPLPYCWLCRALIKRIQAMIPKGALAVAVAQVCRVVP SEQ ID NO: 74 FPIPLPYCWLCRALIKRIQAMIPKGALAVAVAQVCRVV SEQ ID NO: 75 FPIPLPYCWLCRALIKRIQAMIPKGALAVAVAQVCRV SEQ ID NO: 76 FPIPLPYCWLCRALIKRIQAMIPKGALAVAVAQVCR SEQ ID NO: 77 FPIPLPYCWLCRALIKRIQAMIPKGALAVAVAQVC SEQ ID NO: 78 FPIPLPYCWLCRALIKRIQAMIPKGALAVAVAQV SEQ ID NO: 79 FPIPLPYCWLCRALIKRIQAMIPKGALAVAVAQ SEQ ID NO: 80 FPIPLPYCWLCRALIKRIQAMIPKGALAVAVA SEQ ID NO: 81 FPIPLPYCWLCRALIKRIQAMIPKGALAVAV SEQ ID NO: 82 FPIPLPYCWLCRALIKRIQAMIPKGALAVA SEQ ID NO: 83 FPIPLPYCWLCRALIKRIQAMIPKGALAV SEQ ID NO: 84 FPIPLPYCWLCRALIKRIQAMIPKGALA SEQ ID NO: 85 FPIPLPYCWLCRALIKRIQAMIPKGAL SEQ ID NO: 86 FPIPLPYCWLCRALIKRIQAMIPKGA SEQ ID NO: 87 FPIPLPYCWLCRALIKRIQAMIPKG SEQ ID NO: 88 FPIPLPYCWLCRALIKRIQAMIPK SEQ ID NO: 89 FPIPLPYCWLCRALIKRIQAMIP SEQ ID NO: 90 FPIPLPYCWLCRALIKRIQAMI SEQ ID NO: 91 FPIPLPYCWLCRALIKRIQAM SEQ ID NO: 92 FPIPLPYCWLCRALIKRIQA SEQ ID NO: 93 

I claim:
 1. A method of treating a human suffering from or at risk of a lung disease, the method comprising: providing a conjugate comprising a pulmonary active drug for lung treatment selected from the group consisting of an antihistamines; a P2X agonist; a P2Y agonist; a matrix metalloproteinase inhibitors; a kinase inhibitor; an endothelin receptor antagonist; a prostacyclin analogue selected from the group consisting of beraprost, epoprostenol, iloprost, and treprostinil; and a phosphodiesterase type 5 inhibitor; wherein said pulmonary active drug is covalently bonded to a surface active agent characterized by an affinity for the human alveolar/gas interface and comprising at least a portion of a mammalian lung surfactant polypeptide substantially non-immunogenic to humans, wherein the pulmonary active drug binds to an extracellular or cell surface-bound target accessible to the human alveolar/gas interface when the conjugate is present at the interface; and administering the conjugate to the human by inhalation in an amount effective to induce a drug effect in the lungs, wherein said administration increases residence time of the pulmonary active drug in the lung relative to inhalation administration of unconjugated pulmonary active drug.
 2. The method of claim 1 wherein said administration reduces systemic bioavailability of the pulmonary active drug relative to inhalation administration of unconjugated pulmonary active drug.
 3. A method of adapting a pulmonary active drug to improve its lung pharmacodynamic bioavailability comprising: covalently bonding the pulmonary active drug to a surface active agent to produce a pulmonary active drug conjugate, the surface active agent being characterized by an affinity for the human alveolar/gas interface and comprising at least a portion of a mammalian lung surfactant polypeptide, the conjugate having an increased concentration or an increased residence time in the lung relative to the pulmonary active drug administered by inhalation at the same concentration as the conjugate, wherein the pulmonary active drug binds to an extracellular or cell surface-bound target accessible to the human alveolar/gas interface when the conjugate is present at the interface; wherein the pulmonary active drug is selected from the group consisting of: an antihistamine; a P2X agonist; a P2Y agonist; a matrix metalloproteinase inhibitor; a kinase inhibitor; an endothelin receptor antagonist; a prostacyclin analogue selected from the group consisting of beraprost, epoprostenol, iloprost, and treprostinil; and a phosphodiesterase type 5 inhibitor.
 4. The method of claim 1, wherein the administration step is repeated once daily, every other day, every three days, every five days, biweekly, or weekly.
 5. The method of claim 1, wherein the administration step is conducted using an inhalation device.
 6. The method of claim 1, wherein the surface active agent comprises a human lung surfactant polypeptide, a non-human mammalian lung surfactant, or a portion thereof.
 7. The method of claim 6, wherein the surface active agent comprises at least a portion of SP-A, SP-B, SP-C, SP-D, or a mixture thereof.
 8. The method of claim 6, wherein the surface active agent comprises at least a portion of SP-B.
 9. The method of claim 1, wherein the surface active agent comprises or is derived from a mammalian lung surfactant harvested from the lungs of a mammal.
 10. The method of claim 1, wherein said surface active agent comprises a peptide selected from the group consisting of SEQ ID Nos. 36 through
 93. 11. The method of claim 1, wherein the pulmonary active drug is an antihistamine selected from the group consisting of acrivastine, astemizole, azatadine, azelastine, brompheniramine, cetirizine, chlorpheniramine, cyclizine, cyproheptadine, diphehydramine, desloratidine, fexofenadine, hydroxyzine, levocetirizine, loratidine, meclizine, mepyramine, methscopolamine, misolastine, phenidamine, promethazine, pyrilamine, tecastemizole, and terfenadine.
 12. The method of claim 1, wherein the pulmonary active drug is an P2Y agonist selected from the group consisting of denufosol tetrasodium, ATP, and an ATP analogue.
 13. The method of claim 1, wherein the pulmonary active drug is a matrix metalloproteinase inhibitor.
 14. The method of claim 1, wherein the pulmonary active drug is a kinase inhibitor selected from the group consisting of gefitinib and erlotinib.
 15. The method of claim 1, wherein the pulmonary active drug is an endothelin receptor antagonist selected from the group consisting of tezosentan, bosentan, sitaxentan, ambrisentan, and atrasentan.
 16. The method of claim 1, wherein the pulmonary active drug is a phophodiesterase type 5 inhibitor selected from the group consisting of sildenafil, tadalafil, and vardenafil.
 17. The method of claim 1, wherein the surface active agent is covalently attached directly to the pulmonary active drug.
 18. The method of claim 1, wherein the surface active agent is covalently attached to the pulmonary active drug by a linker comprising an amide, ester, urea, carbamate, ether, thioether, disubstituted amine, or trisubstituted amine.
 19. The method of claim 1, wherein the pulmonary active drug is covalently attached to the C-terminus of the mammalian lung surfactant polypeptide of the surface active agent through an amide bond formed through an amino group of the drug and a carboxylic acid group of the surfactant polypeptide.
 20. The method of claim 3, wherein the surface active agent comprises a human lung surfactant polypeptide, a non-human mammalian lung surfactant, or a fraction thereof.
 21. The method of claim 20, wherein the surface active agent comprises at least a portion of SP-A, SP-B, SP-C, SP-D, or a mixture thereof.
 22. The method of claim 20, wherein the surface active agent comprises at least a portion of SP-B.
 23. The method of claim 3, wherein said surface active agent comprises a peptide selected from the group consisting of SEQ ID Nos. 36 through
 93. 24. The method of claim 3, wherein the surface active agent is covalently attached directly to the pulmonary active drug.
 25. The method of claim 3, wherein the surface active agent is covalently attached to the pulmonary active drug by a linker comprising an amide, ester, urea, carbamate, ether, thioether, disubstituted amine, or trisubstituted amine.
 26. The method of claim 3, wherein the pulmonary active drug is covalently attached to the N-terminus of the mammalian lung surfactant polypeptide of the surface active agent through an amide bond formed through a carboxyl group of the drug and an amino group of the surfactant polypeptide.
 27. The method of claim 3, wherein the pulmonary active drug is covalently attached to the C-terminus of the mammalian lung surfactant polypeptide of the surface active agent through an amide bond formed through an amino group of the drug and a carboxylic acid group of the surfactant polypeptide.
 28. The method of claim 3, wherein the pulmonary active drug is covalently attached to the mammalian lung surfactant polypeptide of the surface active agent through a linker attached to an oxygen atom of the hydroxyl group of the drug and a carbonyl-carbon atom of the carboxylic acid group of the surfactant polypeptide. 